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Critical role of gap junction communication, calcium and nitric oxide signaling in bystander responses to focal photodynamic injury.

Calì B, Ceolin S, Ceriani F, Bortolozzi M, Agnellini AH, Zorzi V, Predonzani A, Bronte V, Molon B, Mammano F - Oncotarget (2015)

Bottom Line: Here we show that photosentizer activation in a single cell triggers apoptosis in bystander cancer cells, which are electrically coupled by gap junction channels and support the propagation of a Ca2+ wave initiated in the irradiated cell.The latter also acts as source of nitric oxide (NO) that diffuses to bystander cells, in which NO levels are further increased by a mechanism compatible with Ca(2+)-dependent enzymatic production.Pharmacological blockade of connexin channels significantly reduced the extent of apoptosis in bystander cells, consistent with a critical role played by intercellular communication, Ca2+ and NO in the bystander effects triggered by photodynamic therapy.

View Article: PubMed Central - PubMed

Affiliation: Foundation for Advanced Biomedical Research, Venetian Institute of Molecular Medicine, Padua, Italy.

ABSTRACT
Ionizing and nonionizing radiation affect not only directly targeted cells but also surrounding "bystander" cells. The underlying mechanisms and therapeutic role of bystander responses remain incompletely defined. Here we show that photosentizer activation in a single cell triggers apoptosis in bystander cancer cells, which are electrically coupled by gap junction channels and support the propagation of a Ca2+ wave initiated in the irradiated cell. The latter also acts as source of nitric oxide (NO) that diffuses to bystander cells, in which NO levels are further increased by a mechanism compatible with Ca(2+)-dependent enzymatic production. We detected similar signals in tumors grown in dorsal skinfold chambers applied to live mice. Pharmacological blockade of connexin channels significantly reduced the extent of apoptosis in bystander cells, consistent with a critical role played by intercellular communication, Ca2+ and NO in the bystander effects triggered by photodynamic therapy.

No MeSH data available.


Related in: MedlinePlus

Focal photodynamic injury, i.ephoto−activation of the photosensitizer AlClPc for 60 s in a single cell of a C26GM mouse colon carcinoma cell culture, triggers NO and Ca2+ signals that depart from the irradiated cell and rapidly invade bystander cells; these events are followed by cytochrome c release and widespread cell death. (a) Representative false−color images of simultaneously recorded cytosolic NO (top) and Ca2+ (bottom) concentration changes (Δ) during focal photodynamic injury; the irradiated cell is encased in a white region of interest (ROI); scale bar, 50 μm. (b) Single-cell fluorescence traces obtained as pixel averages from the corresponding (color-matched) ROIs in (a); irradiated cell responses are shown as black traces; the vertical dashed line marks the onset of laser irradiation; ΔNO data were normalized to the corresponding maximal response in the irradiated cell (see Methods); (c) The distance at which bystander cell signals reach 50% of their first peak amplitude is shown as a function of time after the onset of focal photodynamic injury. Data are mean ± s.e.m. from n = 6 cultures; the dashed line is a least square linear fit with a slope of 5.6 μm/s. (d) Cultures were rapidly fixed at shown time points after focal photodynamic injury and immunostained with a cytochrome c antibody and the nuclear counter stain Hoechst; note that images in (d) are from different cultures, whereas those in (a) are all from the same culture; scale bar, 25 μm.
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Figure 1: Focal photodynamic injury, i.ephoto−activation of the photosensitizer AlClPc for 60 s in a single cell of a C26GM mouse colon carcinoma cell culture, triggers NO and Ca2+ signals that depart from the irradiated cell and rapidly invade bystander cells; these events are followed by cytochrome c release and widespread cell death. (a) Representative false−color images of simultaneously recorded cytosolic NO (top) and Ca2+ (bottom) concentration changes (Δ) during focal photodynamic injury; the irradiated cell is encased in a white region of interest (ROI); scale bar, 50 μm. (b) Single-cell fluorescence traces obtained as pixel averages from the corresponding (color-matched) ROIs in (a); irradiated cell responses are shown as black traces; the vertical dashed line marks the onset of laser irradiation; ΔNO data were normalized to the corresponding maximal response in the irradiated cell (see Methods); (c) The distance at which bystander cell signals reach 50% of their first peak amplitude is shown as a function of time after the onset of focal photodynamic injury. Data are mean ± s.e.m. from n = 6 cultures; the dashed line is a least square linear fit with a slope of 5.6 μm/s. (d) Cultures were rapidly fixed at shown time points after focal photodynamic injury and immunostained with a cytochrome c antibody and the nuclear counter stain Hoechst; note that images in (d) are from different cultures, whereas those in (a) are all from the same culture; scale bar, 25 μm.

Mentions: By capturing CuFl and fura-2 fluorescence images in rapid sequence, we determined that AlClPc photo−activation reliably caused elevation (Δ) of cytosolic NO and Ca2+ levels in the irradiated cell. Within seconds, ΔNO and ΔCa2+ signals were detected in all (bystander) cells in the ~300 μm Ø field of view (Figure 1a-c). We obtained similar results in C26GM tumors grown within a dorsal skinfold chamber [39] implanted on BALB/c mice (Supplementary Figure 1), suggesting that the underlying signaling mechanisms are relevant for in vivo photodynamic therapy. We also tested focal photodynamic injury protocol in a different tumor cell line (fibrosarcoma, MCA-203) in vitro, and we obtained qualitatively similar results both for ΔNO and ΔCa2+ signals (Supplementary Figure 2). Immunostaining at different time points after focal photodynamic injury revealed cytochrome c release and cell loss progressing radially from the irradiated cell. The process ensued in near-complete depopulation of the field of view within 24 h following focal photodynamic injury (Figure 1d). ΔCa2+, ΔNO and cytochrome c signals were never detected during or after laser irradiation at 671 nm if AlClPc was omitted from the loading solution (3 out of 3 cultures).


Critical role of gap junction communication, calcium and nitric oxide signaling in bystander responses to focal photodynamic injury.

Calì B, Ceolin S, Ceriani F, Bortolozzi M, Agnellini AH, Zorzi V, Predonzani A, Bronte V, Molon B, Mammano F - Oncotarget (2015)

Focal photodynamic injury, i.ephoto−activation of the photosensitizer AlClPc for 60 s in a single cell of a C26GM mouse colon carcinoma cell culture, triggers NO and Ca2+ signals that depart from the irradiated cell and rapidly invade bystander cells; these events are followed by cytochrome c release and widespread cell death. (a) Representative false−color images of simultaneously recorded cytosolic NO (top) and Ca2+ (bottom) concentration changes (Δ) during focal photodynamic injury; the irradiated cell is encased in a white region of interest (ROI); scale bar, 50 μm. (b) Single-cell fluorescence traces obtained as pixel averages from the corresponding (color-matched) ROIs in (a); irradiated cell responses are shown as black traces; the vertical dashed line marks the onset of laser irradiation; ΔNO data were normalized to the corresponding maximal response in the irradiated cell (see Methods); (c) The distance at which bystander cell signals reach 50% of their first peak amplitude is shown as a function of time after the onset of focal photodynamic injury. Data are mean ± s.e.m. from n = 6 cultures; the dashed line is a least square linear fit with a slope of 5.6 μm/s. (d) Cultures were rapidly fixed at shown time points after focal photodynamic injury and immunostained with a cytochrome c antibody and the nuclear counter stain Hoechst; note that images in (d) are from different cultures, whereas those in (a) are all from the same culture; scale bar, 25 μm.
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Related In: Results  -  Collection

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Figure 1: Focal photodynamic injury, i.ephoto−activation of the photosensitizer AlClPc for 60 s in a single cell of a C26GM mouse colon carcinoma cell culture, triggers NO and Ca2+ signals that depart from the irradiated cell and rapidly invade bystander cells; these events are followed by cytochrome c release and widespread cell death. (a) Representative false−color images of simultaneously recorded cytosolic NO (top) and Ca2+ (bottom) concentration changes (Δ) during focal photodynamic injury; the irradiated cell is encased in a white region of interest (ROI); scale bar, 50 μm. (b) Single-cell fluorescence traces obtained as pixel averages from the corresponding (color-matched) ROIs in (a); irradiated cell responses are shown as black traces; the vertical dashed line marks the onset of laser irradiation; ΔNO data were normalized to the corresponding maximal response in the irradiated cell (see Methods); (c) The distance at which bystander cell signals reach 50% of their first peak amplitude is shown as a function of time after the onset of focal photodynamic injury. Data are mean ± s.e.m. from n = 6 cultures; the dashed line is a least square linear fit with a slope of 5.6 μm/s. (d) Cultures were rapidly fixed at shown time points after focal photodynamic injury and immunostained with a cytochrome c antibody and the nuclear counter stain Hoechst; note that images in (d) are from different cultures, whereas those in (a) are all from the same culture; scale bar, 25 μm.
Mentions: By capturing CuFl and fura-2 fluorescence images in rapid sequence, we determined that AlClPc photo−activation reliably caused elevation (Δ) of cytosolic NO and Ca2+ levels in the irradiated cell. Within seconds, ΔNO and ΔCa2+ signals were detected in all (bystander) cells in the ~300 μm Ø field of view (Figure 1a-c). We obtained similar results in C26GM tumors grown within a dorsal skinfold chamber [39] implanted on BALB/c mice (Supplementary Figure 1), suggesting that the underlying signaling mechanisms are relevant for in vivo photodynamic therapy. We also tested focal photodynamic injury protocol in a different tumor cell line (fibrosarcoma, MCA-203) in vitro, and we obtained qualitatively similar results both for ΔNO and ΔCa2+ signals (Supplementary Figure 2). Immunostaining at different time points after focal photodynamic injury revealed cytochrome c release and cell loss progressing radially from the irradiated cell. The process ensued in near-complete depopulation of the field of view within 24 h following focal photodynamic injury (Figure 1d). ΔCa2+, ΔNO and cytochrome c signals were never detected during or after laser irradiation at 671 nm if AlClPc was omitted from the loading solution (3 out of 3 cultures).

Bottom Line: Here we show that photosentizer activation in a single cell triggers apoptosis in bystander cancer cells, which are electrically coupled by gap junction channels and support the propagation of a Ca2+ wave initiated in the irradiated cell.The latter also acts as source of nitric oxide (NO) that diffuses to bystander cells, in which NO levels are further increased by a mechanism compatible with Ca(2+)-dependent enzymatic production.Pharmacological blockade of connexin channels significantly reduced the extent of apoptosis in bystander cells, consistent with a critical role played by intercellular communication, Ca2+ and NO in the bystander effects triggered by photodynamic therapy.

View Article: PubMed Central - PubMed

Affiliation: Foundation for Advanced Biomedical Research, Venetian Institute of Molecular Medicine, Padua, Italy.

ABSTRACT
Ionizing and nonionizing radiation affect not only directly targeted cells but also surrounding "bystander" cells. The underlying mechanisms and therapeutic role of bystander responses remain incompletely defined. Here we show that photosentizer activation in a single cell triggers apoptosis in bystander cancer cells, which are electrically coupled by gap junction channels and support the propagation of a Ca2+ wave initiated in the irradiated cell. The latter also acts as source of nitric oxide (NO) that diffuses to bystander cells, in which NO levels are further increased by a mechanism compatible with Ca(2+)-dependent enzymatic production. We detected similar signals in tumors grown in dorsal skinfold chambers applied to live mice. Pharmacological blockade of connexin channels significantly reduced the extent of apoptosis in bystander cells, consistent with a critical role played by intercellular communication, Ca2+ and NO in the bystander effects triggered by photodynamic therapy.

No MeSH data available.


Related in: MedlinePlus