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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.


Apoptosis assay. Effect of PPRHs on apoptosis. In the co-culture experiments, either only MCF-7 cells (60,000) were transfected with HpCD47Pr-T, only THP-1 cells (1000) with HpSIRPαI7-T or both MCF-7 and THP-1 cells were transfected with the corresponding PPRH or scrambled PPRHs in controls. As positive control an antibody anti-CD47 was used. Then THP-1 cells were differentiated with 3 ng/ml PMA. 48 h after transfection, apoptosis was measured by Rhodamine method: Cells Rho123-negative and IP-negative were considered as apoptotic cells. Data represent the fold-change in apoptosis relative to MCF-7 cells treated with PMA. *p < 0.05
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Fig6: Apoptosis assay. Effect of PPRHs on apoptosis. In the co-culture experiments, either only MCF-7 cells (60,000) were transfected with HpCD47Pr-T, only THP-1 cells (1000) with HpSIRPαI7-T or both MCF-7 and THP-1 cells were transfected with the corresponding PPRH or scrambled PPRHs in controls. As positive control an antibody anti-CD47 was used. Then THP-1 cells were differentiated with 3 ng/ml PMA. 48 h after transfection, apoptosis was measured by Rhodamine method: Cells Rho123-negative and IP-negative were considered as apoptotic cells. Data represent the fold-change in apoptosis relative to MCF-7 cells treated with PMA. *p < 0.05

Mentions: To associate the dead mechanism to the cytotoxic effect observed in the co-culture, we measured the apoptotic effect of the PPRHs at 100 nM after 48 h of incubation using the rhodamine method (Fig. 6). Co-culture transfected with HpCD47Pr-T and HpSIRPαI1-T provoked a 3-fold increase in apoptosis compared to MCF-7 cells treated with PMA, which was higher than the apoptotic effect triggered by anti-CD47 antibody (1.7-fold) used as positive control. No significant difference was detected in the co-culture control whereas the percentage of apoptotic cells in the co-culture transfected with HpCD47-Sc and HpSIRPα-Sc was twice that of the control MCF-7 cells treated with PMA.Fig. 6


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
Apoptosis assay. Effect of PPRHs on apoptosis. In the co-culture experiments, either only MCF-7 cells (60,000) were transfected with HpCD47Pr-T, only THP-1 cells (1000) with HpSIRPαI7-T or both MCF-7 and THP-1 cells were transfected with the corresponding PPRH or scrambled PPRHs in controls. As positive control an antibody anti-CD47 was used. Then THP-1 cells were differentiated with 3 ng/ml PMA. 48 h after transfection, apoptosis was measured by Rhodamine method: Cells Rho123-negative and IP-negative were considered as apoptotic cells. Data represent the fold-change in apoptosis relative to MCF-7 cells treated with PMA. *p < 0.05
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig6: Apoptosis assay. Effect of PPRHs on apoptosis. In the co-culture experiments, either only MCF-7 cells (60,000) were transfected with HpCD47Pr-T, only THP-1 cells (1000) with HpSIRPαI7-T or both MCF-7 and THP-1 cells were transfected with the corresponding PPRH or scrambled PPRHs in controls. As positive control an antibody anti-CD47 was used. Then THP-1 cells were differentiated with 3 ng/ml PMA. 48 h after transfection, apoptosis was measured by Rhodamine method: Cells Rho123-negative and IP-negative were considered as apoptotic cells. Data represent the fold-change in apoptosis relative to MCF-7 cells treated with PMA. *p < 0.05
Mentions: To associate the dead mechanism to the cytotoxic effect observed in the co-culture, we measured the apoptotic effect of the PPRHs at 100 nM after 48 h of incubation using the rhodamine method (Fig. 6). Co-culture transfected with HpCD47Pr-T and HpSIRPαI1-T provoked a 3-fold increase in apoptosis compared to MCF-7 cells treated with PMA, which was higher than the apoptotic effect triggered by anti-CD47 antibody (1.7-fold) used as positive control. No significant difference was detected in the co-culture control whereas the percentage of apoptotic cells in the co-culture transfected with HpCD47-Sc and HpSIRPα-Sc was twice that of the control MCF-7 cells treated with PMA.Fig. 6

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.