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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 pro-inflammatory cytokines. 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 IL-1β (a), IL-18 (b), IL-6 (c), IL-8 (d), and TNF-α (e) 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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Fig3: mRNA levels of pro-inflammatory cytokines. 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 IL-1β (a), IL-18 (b), IL-6 (c), IL-8 (d), and TNF-α (e) 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: Increased expression of cytokines is one of the phenotypic characteristics of differentiated macrophages [19, 20, 27]. To further investigate if THP-1 cells were able to differentiate to macrophages at 1 and 3 ng/ml PMA, mRNA expression of 5 different pro-inflammatory cytokines (IL-1β, IL-18, IL-6, IL-8, and TNF-α) was analyzed by qRT-PCR (Fig. 3). After the addition of PMA, levels of pro-inflammatory cytokines were increased dose and time dependently relative to untreated THP-1 cells. It is worth noting that the levels of cytokines produced in response to PMA are not the same depending on the culture conditions [18]. IL-1β was elevated in all conditions whereas 3 ng/ml PMA induced the highest expression when treated for 72 h (Fig. 3a). IL-18 (Fig. 3b), IL-6 (Fig. 3c) and IL-8 (Fig. 3d) stimulated at the highest level with 3 ng/ml PMA for 72 h + rest and TNF-α (Fig. 3e) increased 4.4 fold with 3 ng/ml PMA for 72 h. These results suggested that THP-1 cells could be differentiated to macrophages using 3 ng/ml PMA for 72 h and the levels of pro-inflammatory cytokines remained enhanced for the 72 h treatment followed by 48 h resting, which was also reflected in the up-regulated surface markers levels.Fig. 3


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 pro-inflammatory cytokines. 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 IL-1β (a), IL-18 (b), IL-6 (c), IL-8 (d), and TNF-α (e) 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)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037635&req=5

Fig3: mRNA levels of pro-inflammatory cytokines. 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 IL-1β (a), IL-18 (b), IL-6 (c), IL-8 (d), and TNF-α (e) 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: Increased expression of cytokines is one of the phenotypic characteristics of differentiated macrophages [19, 20, 27]. To further investigate if THP-1 cells were able to differentiate to macrophages at 1 and 3 ng/ml PMA, mRNA expression of 5 different pro-inflammatory cytokines (IL-1β, IL-18, IL-6, IL-8, and TNF-α) was analyzed by qRT-PCR (Fig. 3). After the addition of PMA, levels of pro-inflammatory cytokines were increased dose and time dependently relative to untreated THP-1 cells. It is worth noting that the levels of cytokines produced in response to PMA are not the same depending on the culture conditions [18]. IL-1β was elevated in all conditions whereas 3 ng/ml PMA induced the highest expression when treated for 72 h (Fig. 3a). IL-18 (Fig. 3b), IL-6 (Fig. 3c) and IL-8 (Fig. 3d) stimulated at the highest level with 3 ng/ml PMA for 72 h + rest and TNF-α (Fig. 3e) increased 4.4 fold with 3 ng/ml PMA for 72 h. These results suggested that THP-1 cells could be differentiated to macrophages using 3 ng/ml PMA for 72 h and the levels of pro-inflammatory cytokines remained enhanced for the 72 h treatment followed by 48 h resting, which was also reflected in the up-regulated surface markers levels.Fig. 3

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.