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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

Summary of pharmacological interference experimentsCells were incubated for 15−20 min with shown drugs prior to focal photodynamic injury; concentrations: EGTA, 100 μM (in nominally Ca2+−free medium); CBX, 100μM; FFA, 100 μM; 2-APB, 100 μM; CPA, 30 μM; suramin (Sur), 200 μM; L-NIO, 10 μM. Data in (a−b) are mean ± s.e.m. of signals measured in 36 randomly selected bystander cells located in an annular region at the periphery of the field of view (between 75 μm and 120 μm from the irradiated cell). (a) NO level change (ΔNO) normalized to the average change measured in control conditions (Ctrl). (b) Cytosolic Ca2+ concentration change (ΔCa2+). (c) Speed of Ca2+ wave elicited by focal photodynamic injury.
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Figure 5: Summary of pharmacological interference experimentsCells were incubated for 15−20 min with shown drugs prior to focal photodynamic injury; concentrations: EGTA, 100 μM (in nominally Ca2+−free medium); CBX, 100μM; FFA, 100 μM; 2-APB, 100 μM; CPA, 30 μM; suramin (Sur), 200 μM; L-NIO, 10 μM. Data in (a−b) are mean ± s.e.m. of signals measured in 36 randomly selected bystander cells located in an annular region at the periphery of the field of view (between 75 μm and 120 μm from the irradiated cell). (a) NO level change (ΔNO) normalized to the average change measured in control conditions (Ctrl). (b) Cytosolic Ca2+ concentration change (ΔCa2+). (c) Speed of Ca2+ wave elicited by focal photodynamic injury.

Mentions: Figure 5 illustrates the effects of gap junction inhibitors and other drugs on the speed of the intercellular Ca2+ wave, the ΔCa2+ and ΔNO signals evoked in bystander cells by focal photodynamic injury. Both CBX and flufenamic acid (FFA), another commonly used, non-specific inhibitor of connexin-made channels [49], caused a significant reductions of these three parameters. We also examined the consequences of perturbing Ca2+ homeostasis prior to focal photodynamic injury. The mild inhibition we observed in Ca2+−free extracellular medium (EGTA) implies negligible contribution of Ca2+ entry to bystander responses. Conversely, all three parameters were significantly reduced if ER Ca2+ levels were lowered by incubating C26GM cultures with cyclopiazonic acid [53] (CPA), a specific inhibitor of sarco/endoplasmic reticulum Ca2+−ATPase (SERCA pumps), in Ca2+ free medium. The most pronounced reductions of ΔNO and Ca2+ bystander signals were obtained with 2APB, a non-specific inhibitor of IP3 receptors (IP3R) [54]. ΔNO and ΔCa2+ signals and the speed of the intercellular Ca2+ wave were significantly attenuated also by suramin [55], suggesting that paracrine signaling mediated by ATP may play a role. Finally, the potent irreversible NOS inhibitor N-iminoethyl-L-ornithine (L-NIO) [56] significantly reduced bystander NO responses (Figure 5a) without affecting bystander Ca2+ signaling (Figure 5b, C).


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)

Summary of pharmacological interference experimentsCells were incubated for 15−20 min with shown drugs prior to focal photodynamic injury; concentrations: EGTA, 100 μM (in nominally Ca2+−free medium); CBX, 100μM; FFA, 100 μM; 2-APB, 100 μM; CPA, 30 μM; suramin (Sur), 200 μM; L-NIO, 10 μM. Data in (a−b) are mean ± s.e.m. of signals measured in 36 randomly selected bystander cells located in an annular region at the periphery of the field of view (between 75 μm and 120 μm from the irradiated cell). (a) NO level change (ΔNO) normalized to the average change measured in control conditions (Ctrl). (b) Cytosolic Ca2+ concentration change (ΔCa2+). (c) Speed of Ca2+ wave elicited by focal photodynamic injury.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Summary of pharmacological interference experimentsCells were incubated for 15−20 min with shown drugs prior to focal photodynamic injury; concentrations: EGTA, 100 μM (in nominally Ca2+−free medium); CBX, 100μM; FFA, 100 μM; 2-APB, 100 μM; CPA, 30 μM; suramin (Sur), 200 μM; L-NIO, 10 μM. Data in (a−b) are mean ± s.e.m. of signals measured in 36 randomly selected bystander cells located in an annular region at the periphery of the field of view (between 75 μm and 120 μm from the irradiated cell). (a) NO level change (ΔNO) normalized to the average change measured in control conditions (Ctrl). (b) Cytosolic Ca2+ concentration change (ΔCa2+). (c) Speed of Ca2+ wave elicited by focal photodynamic injury.
Mentions: Figure 5 illustrates the effects of gap junction inhibitors and other drugs on the speed of the intercellular Ca2+ wave, the ΔCa2+ and ΔNO signals evoked in bystander cells by focal photodynamic injury. Both CBX and flufenamic acid (FFA), another commonly used, non-specific inhibitor of connexin-made channels [49], caused a significant reductions of these three parameters. We also examined the consequences of perturbing Ca2+ homeostasis prior to focal photodynamic injury. The mild inhibition we observed in Ca2+−free extracellular medium (EGTA) implies negligible contribution of Ca2+ entry to bystander responses. Conversely, all three parameters were significantly reduced if ER Ca2+ levels were lowered by incubating C26GM cultures with cyclopiazonic acid [53] (CPA), a specific inhibitor of sarco/endoplasmic reticulum Ca2+−ATPase (SERCA pumps), in Ca2+ free medium. The most pronounced reductions of ΔNO and Ca2+ bystander signals were obtained with 2APB, a non-specific inhibitor of IP3 receptors (IP3R) [54]. ΔNO and ΔCa2+ signals and the speed of the intercellular Ca2+ wave were significantly attenuated also by suramin [55], suggesting that paracrine signaling mediated by ATP may play a role. Finally, the potent irreversible NOS inhibitor N-iminoethyl-L-ornithine (L-NIO) [56] significantly reduced bystander NO responses (Figure 5a) without affecting bystander Ca2+ signaling (Figure 5b, C).

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