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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) Viability of EMT-6 cells as determined 24 h after photodynamic therapy (PDT) with zinc phthalocyanine-encapsulating endothelium-targeted liposomes (ZnPC-ETLs) at the indicated concentrations. Cells were placed in hypoxic conditions immediately following PDT (mean ± SD, N = 6). Data were analyzed using a one-way ANOVA and Sidak’s post-hoc test. Significant differences versus the corresponding control group are indicated with a pound sign, whereas are indicated with asterisks. The number of signs indicates the level of significance: *p < 0.05, ****p < 0.001 for intergroup differences, #### p < 0.001 versus control; (B) Flow cytometry-based characterization of the mode of EMT-6 cell death 24 h after PDT and subsequent incubation under hypoxic conditions (mean ± SD, N = 3). Staining was performed with Alexa Fluor 488-conjugated annexin V (apoptosis) and propidium iodide (necrosis); (C) The medium from EMT-6 cells was harvested 24 h after PDT and subsequent hypoxic incubation, and added to cultured RAW 264.7 macrophages. After 24 h of stimulation, macrophage activation was assessed by measuring nitric oxide (NO) production, which is a measure of macrophage activation. Cells were stimulated with 1 μg/mL lipopolysaccharide (LPS) for 24 h as positive control. The number behind “PDT” refers to the final lipid concentration of ZnPC-ETLs; (D–G) The medium from PDT-treated EMT-6 cells was analyzed for healthy, apoptotic (green), and necrotic (red) cells by flow cytometry; (H–K) Cytokine release by PDT-treated EMT-6 cells was assayed 24 h after PDT and subsequent hypoxic incubation (mean ± SD, N = 3). The “0 μM ZnPC-ETLs” group was not irradiated and served as negative control. Data were analyzed using a Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparisons. The ZnPC-ETL concentrations (x-axes) refer to final lipid concentration. The ZnPC:lipid molar ratio was 0.003 [6]. *p < 0.05.
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ijms-16-19960-f001: (A) Viability of EMT-6 cells as determined 24 h after photodynamic therapy (PDT) with zinc phthalocyanine-encapsulating endothelium-targeted liposomes (ZnPC-ETLs) at the indicated concentrations. Cells were placed in hypoxic conditions immediately following PDT (mean ± SD, N = 6). Data were analyzed using a one-way ANOVA and Sidak’s post-hoc test. Significant differences versus the corresponding control group are indicated with a pound sign, whereas are indicated with asterisks. The number of signs indicates the level of significance: *p < 0.05, ****p < 0.001 for intergroup differences, #### p < 0.001 versus control; (B) Flow cytometry-based characterization of the mode of EMT-6 cell death 24 h after PDT and subsequent incubation under hypoxic conditions (mean ± SD, N = 3). Staining was performed with Alexa Fluor 488-conjugated annexin V (apoptosis) and propidium iodide (necrosis); (C) The medium from EMT-6 cells was harvested 24 h after PDT and subsequent hypoxic incubation, and added to cultured RAW 264.7 macrophages. After 24 h of stimulation, macrophage activation was assessed by measuring nitric oxide (NO) production, which is a measure of macrophage activation. Cells were stimulated with 1 μg/mL lipopolysaccharide (LPS) for 24 h as positive control. The number behind “PDT” refers to the final lipid concentration of ZnPC-ETLs; (D–G) The medium from PDT-treated EMT-6 cells was analyzed for healthy, apoptotic (green), and necrotic (red) cells by flow cytometry; (H–K) Cytokine release by PDT-treated EMT-6 cells was assayed 24 h after PDT and subsequent hypoxic incubation (mean ± SD, N = 3). The “0 μM ZnPC-ETLs” group was not irradiated and served as negative control. Data were analyzed using a Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparisons. The ZnPC-ETL concentrations (x-axes) refer to final lipid concentration. The ZnPC:lipid molar ratio was 0.003 [6]. *p < 0.05.

Mentions: First, the PDT efficacy of ZnPC-ETLs was determined in EMT-6 cells at 24 h post-PDT and subsequent hypoxic incubation, showing a significant photosensitizer dose-dependent decrease in viability (Figure 1A). Flow cytometric analysis performed 24 h post-PDT revealed that tumor cell death occurred predominantly via apoptosis and potentially secondary necrosis (Figure 1B). The addition of PDT-treated EMT-6 cell supernatant to subconfluent RAW 264.7 macrophages caused an increase in nitric oxide (NO) production (Figure 1C), which is a hallmark of macrophage activation [27]. The supernatant of PDT-subjected EMT-6 cells contained viable, apoptotic, and necrotic cells. The quantity of the supernatant-suspended cells increased in a ZnPC concentration-dependent manner (Figure 1D–G), which was inversely proportional to post-PDT cell viability (Figure 1A). Moreover, the supernatant of EMT-6 cells subjected to PDT at the highest ZnPC dose induced the most extensive macrophage activation (Figure 1C), demonstrating that PDT triggers innate immune cell activation in proportion to the degree of tumor cell injury.


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) Viability of EMT-6 cells as determined 24 h after photodynamic therapy (PDT) with zinc phthalocyanine-encapsulating endothelium-targeted liposomes (ZnPC-ETLs) at the indicated concentrations. Cells were placed in hypoxic conditions immediately following PDT (mean ± SD, N = 6). Data were analyzed using a one-way ANOVA and Sidak’s post-hoc test. Significant differences versus the corresponding control group are indicated with a pound sign, whereas are indicated with asterisks. The number of signs indicates the level of significance: *p < 0.05, ****p < 0.001 for intergroup differences, #### p < 0.001 versus control; (B) Flow cytometry-based characterization of the mode of EMT-6 cell death 24 h after PDT and subsequent incubation under hypoxic conditions (mean ± SD, N = 3). Staining was performed with Alexa Fluor 488-conjugated annexin V (apoptosis) and propidium iodide (necrosis); (C) The medium from EMT-6 cells was harvested 24 h after PDT and subsequent hypoxic incubation, and added to cultured RAW 264.7 macrophages. After 24 h of stimulation, macrophage activation was assessed by measuring nitric oxide (NO) production, which is a measure of macrophage activation. Cells were stimulated with 1 μg/mL lipopolysaccharide (LPS) for 24 h as positive control. The number behind “PDT” refers to the final lipid concentration of ZnPC-ETLs; (D–G) The medium from PDT-treated EMT-6 cells was analyzed for healthy, apoptotic (green), and necrotic (red) cells by flow cytometry; (H–K) Cytokine release by PDT-treated EMT-6 cells was assayed 24 h after PDT and subsequent hypoxic incubation (mean ± SD, N = 3). The “0 μM ZnPC-ETLs” group was not irradiated and served as negative control. Data were analyzed using a Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparisons. The ZnPC-ETL concentrations (x-axes) refer to final lipid concentration. The ZnPC:lipid molar ratio was 0.003 [6]. *p < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-19960-f001: (A) Viability of EMT-6 cells as determined 24 h after photodynamic therapy (PDT) with zinc phthalocyanine-encapsulating endothelium-targeted liposomes (ZnPC-ETLs) at the indicated concentrations. Cells were placed in hypoxic conditions immediately following PDT (mean ± SD, N = 6). Data were analyzed using a one-way ANOVA and Sidak’s post-hoc test. Significant differences versus the corresponding control group are indicated with a pound sign, whereas are indicated with asterisks. The number of signs indicates the level of significance: *p < 0.05, ****p < 0.001 for intergroup differences, #### p < 0.001 versus control; (B) Flow cytometry-based characterization of the mode of EMT-6 cell death 24 h after PDT and subsequent incubation under hypoxic conditions (mean ± SD, N = 3). Staining was performed with Alexa Fluor 488-conjugated annexin V (apoptosis) and propidium iodide (necrosis); (C) The medium from EMT-6 cells was harvested 24 h after PDT and subsequent hypoxic incubation, and added to cultured RAW 264.7 macrophages. After 24 h of stimulation, macrophage activation was assessed by measuring nitric oxide (NO) production, which is a measure of macrophage activation. Cells were stimulated with 1 μg/mL lipopolysaccharide (LPS) for 24 h as positive control. The number behind “PDT” refers to the final lipid concentration of ZnPC-ETLs; (D–G) The medium from PDT-treated EMT-6 cells was analyzed for healthy, apoptotic (green), and necrotic (red) cells by flow cytometry; (H–K) Cytokine release by PDT-treated EMT-6 cells was assayed 24 h after PDT and subsequent hypoxic incubation (mean ± SD, N = 3). The “0 μM ZnPC-ETLs” group was not irradiated and served as negative control. Data were analyzed using a Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparisons. The ZnPC-ETL concentrations (x-axes) refer to final lipid concentration. The ZnPC:lipid molar ratio was 0.003 [6]. *p < 0.05.
Mentions: First, the PDT efficacy of ZnPC-ETLs was determined in EMT-6 cells at 24 h post-PDT and subsequent hypoxic incubation, showing a significant photosensitizer dose-dependent decrease in viability (Figure 1A). Flow cytometric analysis performed 24 h post-PDT revealed that tumor cell death occurred predominantly via apoptosis and potentially secondary necrosis (Figure 1B). The addition of PDT-treated EMT-6 cell supernatant to subconfluent RAW 264.7 macrophages caused an increase in nitric oxide (NO) production (Figure 1C), which is a hallmark of macrophage activation [27]. The supernatant of PDT-subjected EMT-6 cells contained viable, apoptotic, and necrotic cells. The quantity of the supernatant-suspended cells increased in a ZnPC concentration-dependent manner (Figure 1D–G), which was inversely proportional to post-PDT cell viability (Figure 1A). Moreover, the supernatant of EMT-6 cells subjected to PDT at the highest ZnPC dose induced the most extensive macrophage activation (Figure 1C), demonstrating that PDT triggers innate immune cell activation in proportion to the degree of tumor cell injury.

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