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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

Comparison of experimental and model responses highlights dual contribution to NO signaling in bystander cells(a) Experimental ΔNO traces evoked by focal photodynamic injury at increasing distances from the irradiated cell (black solid line). (b) ΔNO signals in bystander cells predicted by a purely diffusive model using the irradiated cell signal in (a) as input and a diffusion coefficient DNO = 3300 μm2/s. (c) Differences between the traces shown in (a) and (b), which we interpret as enzymatic contributions to bystander responses. (d) Maximal measured and diffusive NO level increments (ΔNOmax) in bystander cells vs. distance from the irradiated cell. (e) Ratio of enzymatic ΔNOmax over diffusive ΔNOmax vs. distance from the irradiated cell. Measured data in (d) and (e) are mean ± s.e.m. from n = 3 cultures; those in (d) were normalized to the corresponding maximal response in the irradiated cell.
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Figure 3: Comparison of experimental and model responses highlights dual contribution to NO signaling in bystander cells(a) Experimental ΔNO traces evoked by focal photodynamic injury at increasing distances from the irradiated cell (black solid line). (b) ΔNO signals in bystander cells predicted by a purely diffusive model using the irradiated cell signal in (a) as input and a diffusion coefficient DNO = 3300 μm2/s. (c) Differences between the traces shown in (a) and (b), which we interpret as enzymatic contributions to bystander responses. (d) Maximal measured and diffusive NO level increments (ΔNOmax) in bystander cells vs. distance from the irradiated cell. (e) Ratio of enzymatic ΔNOmax over diffusive ΔNOmax vs. distance from the irradiated cell. Measured data in (d) and (e) are mean ± s.e.m. from n = 3 cultures; those in (d) were normalized to the corresponding maximal response in the irradiated cell.

Mentions: To get deeper insight into the intracellular and intercellular dynamics of ΔNO signals evoked by focal photodynamic injury, we created a mathematical model (see Methods, Equation 2, Supplementary Methods and Supplementary Figure 3) assuming that NO: (i) is generated within and released from the irradiated cell; (ii) diffuses freely across the extracellular space; (iii) passes freely through cell membranes of bystander cells, in which it is finally detected by pre−loaded CuFl. We used one of the ΔNO traces measured in an irradiated cell as input to this model and computed ΔNO bystander responses. The results of this analysis (Figure 3) show that ΔNO responses measured in bystander cells (Figure 3a) largely exceed those predicted based solely on NO diffusion (Figure 3b). The differences between measured and diffusive ΔNO signals provide estimates of the alternative generation of NO in bystander cells, likely by its enzymatic production by NOS (Figure 3c). Both the measured NO level increments and the purely diffusive component (estimated by the mathematical model) are monotonically decreasing functions of distance from the irradiated cell (Figure 3d), however the diffusive contribution exhibits a faster spatial rate of decrease. Consequently the ratio of measured minus diffusive (i.e. enzymatic) ΔNOmax over diffusive ΔNOmax shows a tendency to increase towards the periphery of the field of view, where it is >2 (Figure 3e).


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)

Comparison of experimental and model responses highlights dual contribution to NO signaling in bystander cells(a) Experimental ΔNO traces evoked by focal photodynamic injury at increasing distances from the irradiated cell (black solid line). (b) ΔNO signals in bystander cells predicted by a purely diffusive model using the irradiated cell signal in (a) as input and a diffusion coefficient DNO = 3300 μm2/s. (c) Differences between the traces shown in (a) and (b), which we interpret as enzymatic contributions to bystander responses. (d) Maximal measured and diffusive NO level increments (ΔNOmax) in bystander cells vs. distance from the irradiated cell. (e) Ratio of enzymatic ΔNOmax over diffusive ΔNOmax vs. distance from the irradiated cell. Measured data in (d) and (e) are mean ± s.e.m. from n = 3 cultures; those in (d) were normalized to the corresponding maximal response in the irradiated cell.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496347&req=5

Figure 3: Comparison of experimental and model responses highlights dual contribution to NO signaling in bystander cells(a) Experimental ΔNO traces evoked by focal photodynamic injury at increasing distances from the irradiated cell (black solid line). (b) ΔNO signals in bystander cells predicted by a purely diffusive model using the irradiated cell signal in (a) as input and a diffusion coefficient DNO = 3300 μm2/s. (c) Differences between the traces shown in (a) and (b), which we interpret as enzymatic contributions to bystander responses. (d) Maximal measured and diffusive NO level increments (ΔNOmax) in bystander cells vs. distance from the irradiated cell. (e) Ratio of enzymatic ΔNOmax over diffusive ΔNOmax vs. distance from the irradiated cell. Measured data in (d) and (e) are mean ± s.e.m. from n = 3 cultures; those in (d) were normalized to the corresponding maximal response in the irradiated cell.
Mentions: To get deeper insight into the intracellular and intercellular dynamics of ΔNO signals evoked by focal photodynamic injury, we created a mathematical model (see Methods, Equation 2, Supplementary Methods and Supplementary Figure 3) assuming that NO: (i) is generated within and released from the irradiated cell; (ii) diffuses freely across the extracellular space; (iii) passes freely through cell membranes of bystander cells, in which it is finally detected by pre−loaded CuFl. We used one of the ΔNO traces measured in an irradiated cell as input to this model and computed ΔNO bystander responses. The results of this analysis (Figure 3) show that ΔNO responses measured in bystander cells (Figure 3a) largely exceed those predicted based solely on NO diffusion (Figure 3b). The differences between measured and diffusive ΔNO signals provide estimates of the alternative generation of NO in bystander cells, likely by its enzymatic production by NOS (Figure 3c). Both the measured NO level increments and the purely diffusive component (estimated by the mathematical model) are monotonically decreasing functions of distance from the irradiated cell (Figure 3d), however the diffusive contribution exhibits a faster spatial rate of decrease. Consequently the ratio of measured minus diffusive (i.e. enzymatic) ΔNOmax over diffusive ΔNOmax shows a tendency to increase towards the periphery of the field of view, where it is >2 (Figure 3e).

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