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Inhibition of NF-κB in Tumor Cells Exacerbates Immune Cell Activation Following Photodynamic Therapy.

Broekgaarden M, Kos M, Jurg FA, van Beek AA, van Gulik TM, Heger M - Int J Mol Sci (2015)

Bottom Line: One of these strategies is to combine PDT with inhibitors of PDT-induced survival pathways.In contrast to these postulations, this study demonstrated that siRNA knockdown of NF-κB in murine breast carcinoma (EMT-6) cells increased survival signaling in these cells and exacerbated the inflammatory response in murine RAW 264.7 macrophages.These results suggest a pro-death and immunosuppressive role of NF-κB in PDT-treated cells that concurs with a hyperstimulated immune response in innate immune cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Surgery, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands. mbroekgaarden@mgh.harvard.edu.

ABSTRACT
Although photodynamic therapy (PDT) yields very good outcomes in numerous types of superficial solid cancers, some tumors respond suboptimally to PDT. Novel treatment strategies are therefore needed to enhance the efficacy in these therapy-resistant tumors. One of these strategies is to combine PDT with inhibitors of PDT-induced survival pathways. In this respect, the transcription factor nuclear factor κB (NF-κB) has been identified as a potential pharmacological target, albeit inhibition of NF-κB may concurrently dampen the subsequent anti-tumor immune response required for complete tumor eradication and abscopal effects. In contrast to these postulations, this study demonstrated that siRNA knockdown of NF-κB in murine breast carcinoma (EMT-6) cells increased survival signaling in these cells and exacerbated the inflammatory response in murine RAW 264.7 macrophages. These results suggest a pro-death and immunosuppressive role of NF-κB in PDT-treated cells that concurs with a hyperstimulated immune response in innate immune cells.

No MeSH data available.


Related in: MedlinePlus

(A) EMT-6 cells were treated with either sham siRNA (black bars) or RelA siRNA (grey bars) and subsequently subjected to PDT with increasing concentrations of ZnPC-ETLs (indicated by the number after “PDT”). Cell viability was assessed 24 h after PDT and subsequent hypoxic incubation (mean ± SD, N = 6, student’s t-test). ***p < 0.005; (B) ZnPC-ETL IC50 values as determined on EMT-6 cells that were preconditioned with LPS versus non-preconditioned cells. ***p < 0.005; (C) NO production by RAW 264.7 macrophages 24 h after stimulation with supernatant from PDT-treated EMT-6 or EMT-6-RelAkd cells (mean ± SD, N = 3); (D–I) After 24 h of incubation with supernatant from PDT-treated EMT-6 or EMT-6-RelAkd cells, the medium from the RAW 264.7 cells was assayed for cytokine levels (means ± SD, N = 3). The ZnPC-ETL concentrations (x-axes) refer to final lipid concentration (indicated by the number after “PDT”). The ZnPC:lipid molar ratio was 0.003 [6].
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ijms-16-19960-f002: (A) EMT-6 cells were treated with either sham siRNA (black bars) or RelA siRNA (grey bars) and subsequently subjected to PDT with increasing concentrations of ZnPC-ETLs (indicated by the number after “PDT”). Cell viability was assessed 24 h after PDT and subsequent hypoxic incubation (mean ± SD, N = 6, student’s t-test). ***p < 0.005; (B) ZnPC-ETL IC50 values as determined on EMT-6 cells that were preconditioned with LPS versus non-preconditioned cells. ***p < 0.005; (C) NO production by RAW 264.7 macrophages 24 h after stimulation with supernatant from PDT-treated EMT-6 or EMT-6-RelAkd cells (mean ± SD, N = 3); (D–I) After 24 h of incubation with supernatant from PDT-treated EMT-6 or EMT-6-RelAkd cells, the medium from the RAW 264.7 cells was assayed for cytokine levels (means ± SD, N = 3). The ZnPC-ETL concentrations (x-axes) refer to final lipid concentration (indicated by the number after “PDT”). The ZnPC:lipid molar ratio was 0.003 [6].

Mentions: Given that NF-κB mediates cell survival [9,10] as well as transcriptional upregulation and synthesis of the assayed cytokines [2,13], it was hypothesized that inhibition of NF-κB in EMT-6 cells would improve PDT efficacy and reduce the pro-inflammatory signaling by PDT-afflicted tumor cells. Accordingly, the mRNA that encodes RelA (reticuloendotheliosis A, nuclear factor NF-κB p65 subunit) was knocked down with siRNA (designated as “EMT-6-RelAkd”), thereby abrogating the activity of an essential subunit of the NF-κB transcription factors [30]. Control cells were transfected with sham siRNA. The EMT-6-RelAkd cells were subsequently subjected to the same PDT protocol as used in the previous assays (Figure 1). EMT-6-RelAkd cells were more resistant to PDT-induced cell death (Figure 2A), indicating that NF-κB plays a pivotal role in the promotion of EMT-6 tumor cell death following PDT. As NF-κB can be activated with lipopolysaccharide (LPS) [31,32], the results were corroborated in positive control experiments. Tumor cells pretreated with LPS were significantly more susceptible to PDT compared to non-pretreated cells (Figure 2B).


Inhibition of NF-κB in Tumor Cells Exacerbates Immune Cell Activation Following Photodynamic Therapy.

Broekgaarden M, Kos M, Jurg FA, van Beek AA, van Gulik TM, Heger M - Int J Mol Sci (2015)

(A) EMT-6 cells were treated with either sham siRNA (black bars) or RelA siRNA (grey bars) and subsequently subjected to PDT with increasing concentrations of ZnPC-ETLs (indicated by the number after “PDT”). Cell viability was assessed 24 h after PDT and subsequent hypoxic incubation (mean ± SD, N = 6, student’s t-test). ***p < 0.005; (B) ZnPC-ETL IC50 values as determined on EMT-6 cells that were preconditioned with LPS versus non-preconditioned cells. ***p < 0.005; (C) NO production by RAW 264.7 macrophages 24 h after stimulation with supernatant from PDT-treated EMT-6 or EMT-6-RelAkd cells (mean ± SD, N = 3); (D–I) After 24 h of incubation with supernatant from PDT-treated EMT-6 or EMT-6-RelAkd cells, the medium from the RAW 264.7 cells was assayed for cytokine levels (means ± SD, N = 3). The ZnPC-ETL concentrations (x-axes) refer to final lipid concentration (indicated by the number after “PDT”). The ZnPC:lipid molar ratio was 0.003 [6].
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581334&req=5

ijms-16-19960-f002: (A) EMT-6 cells were treated with either sham siRNA (black bars) or RelA siRNA (grey bars) and subsequently subjected to PDT with increasing concentrations of ZnPC-ETLs (indicated by the number after “PDT”). Cell viability was assessed 24 h after PDT and subsequent hypoxic incubation (mean ± SD, N = 6, student’s t-test). ***p < 0.005; (B) ZnPC-ETL IC50 values as determined on EMT-6 cells that were preconditioned with LPS versus non-preconditioned cells. ***p < 0.005; (C) NO production by RAW 264.7 macrophages 24 h after stimulation with supernatant from PDT-treated EMT-6 or EMT-6-RelAkd cells (mean ± SD, N = 3); (D–I) After 24 h of incubation with supernatant from PDT-treated EMT-6 or EMT-6-RelAkd cells, the medium from the RAW 264.7 cells was assayed for cytokine levels (means ± SD, N = 3). The ZnPC-ETL concentrations (x-axes) refer to final lipid concentration (indicated by the number after “PDT”). The ZnPC:lipid molar ratio was 0.003 [6].
Mentions: Given that NF-κB mediates cell survival [9,10] as well as transcriptional upregulation and synthesis of the assayed cytokines [2,13], it was hypothesized that inhibition of NF-κB in EMT-6 cells would improve PDT efficacy and reduce the pro-inflammatory signaling by PDT-afflicted tumor cells. Accordingly, the mRNA that encodes RelA (reticuloendotheliosis A, nuclear factor NF-κB p65 subunit) was knocked down with siRNA (designated as “EMT-6-RelAkd”), thereby abrogating the activity of an essential subunit of the NF-κB transcription factors [30]. Control cells were transfected with sham siRNA. The EMT-6-RelAkd cells were subsequently subjected to the same PDT protocol as used in the previous assays (Figure 1). EMT-6-RelAkd cells were more resistant to PDT-induced cell death (Figure 2A), indicating that NF-κB plays a pivotal role in the promotion of EMT-6 tumor cell death following PDT. As NF-κB can be activated with lipopolysaccharide (LPS) [31,32], the results were corroborated in positive control experiments. Tumor cells pretreated with LPS were significantly more susceptible to PDT compared to non-pretreated cells (Figure 2B).

Bottom Line: One of these strategies is to combine PDT with inhibitors of PDT-induced survival pathways.In contrast to these postulations, this study demonstrated that siRNA knockdown of NF-κB in murine breast carcinoma (EMT-6) cells increased survival signaling in these cells and exacerbated the inflammatory response in murine RAW 264.7 macrophages.These results suggest a pro-death and immunosuppressive role of NF-κB in PDT-treated cells that concurs with a hyperstimulated immune response in innate immune cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Surgery, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands. mbroekgaarden@mgh.harvard.edu.

ABSTRACT
Although photodynamic therapy (PDT) yields very good outcomes in numerous types of superficial solid cancers, some tumors respond suboptimally to PDT. Novel treatment strategies are therefore needed to enhance the efficacy in these therapy-resistant tumors. One of these strategies is to combine PDT with inhibitors of PDT-induced survival pathways. In this respect, the transcription factor nuclear factor κB (NF-κB) has been identified as a potential pharmacological target, albeit inhibition of NF-κB may concurrently dampen the subsequent anti-tumor immune response required for complete tumor eradication and abscopal effects. In contrast to these postulations, this study demonstrated that siRNA knockdown of NF-κB in murine breast carcinoma (EMT-6) cells increased survival signaling in these cells and exacerbated the inflammatory response in murine RAW 264.7 macrophages. These results suggest a pro-death and immunosuppressive role of NF-κB in PDT-treated cells that concurs with a hyperstimulated immune response in innate immune cells.

No MeSH data available.


Related in: MedlinePlus