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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

C26GM cells are coupled by gap junction channels(a) Coupling assay based on voltage imaging with the Vf.2.1.Cl membrane potential sensor shows cells are coupled by gap-junction channels (Ctrl), which can be blocked by carbenoxolone (CBX, 100 μM); scale bar, 50 μm. (b) Relative abundance of connexin transcripts in C26GM cultured cells assayed by qPCR.
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Figure 4: C26GM cells are coupled by gap junction channels(a) Coupling assay based on voltage imaging with the Vf.2.1.Cl membrane potential sensor shows cells are coupled by gap-junction channels (Ctrl), which can be blocked by carbenoxolone (CBX, 100 μM); scale bar, 50 μm. (b) Relative abundance of connexin transcripts in C26GM cultured cells assayed by qPCR.

Mentions: To test these hypotheses we performed a series of pharmacological interference experiments. We noted that the relatively low value of the Ca2+ wave speed is compatible with a propagation mechanism whereby diffusion of soluble messengers, such as IP3, through gap junction channels plays a significant role [40]. Gap junction communication has been repeatedly implicated in bystander responses to ionizing radiation [41-46]. Therefore, we assayed C26GM cultures for the presence of functional intercellular channels using a novel, highly sensitive approach [47], based on a combination of patch clamp and voltage imaging with the membrane potential reporter Vf.2.1.Cl [48]. Our results (Figure 4a) indicate that cultured C26GM cells form functional syncytia since (i) electrical signals delivered to the patch-clamped cell invaded a number of other cells in the culture and (ii) electrical coupling was reversibly abrogated by carbenoxolone (CBX), a widely used non-specific inhibitor of connexin-made channels [49]. qPCR analysis for five different connexins expressed in various tumors [50-52] singled out Cx43 as the predominant isoform expressed by C26GM cells, whereas Cx40 and Cx26 provide minor contributions (Figure 4b).


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)

C26GM cells are coupled by gap junction channels(a) Coupling assay based on voltage imaging with the Vf.2.1.Cl membrane potential sensor shows cells are coupled by gap-junction channels (Ctrl), which can be blocked by carbenoxolone (CBX, 100 μM); scale bar, 50 μm. (b) Relative abundance of connexin transcripts in C26GM cultured cells assayed by qPCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4496347&req=5

Figure 4: C26GM cells are coupled by gap junction channels(a) Coupling assay based on voltage imaging with the Vf.2.1.Cl membrane potential sensor shows cells are coupled by gap-junction channels (Ctrl), which can be blocked by carbenoxolone (CBX, 100 μM); scale bar, 50 μm. (b) Relative abundance of connexin transcripts in C26GM cultured cells assayed by qPCR.
Mentions: To test these hypotheses we performed a series of pharmacological interference experiments. We noted that the relatively low value of the Ca2+ wave speed is compatible with a propagation mechanism whereby diffusion of soluble messengers, such as IP3, through gap junction channels plays a significant role [40]. Gap junction communication has been repeatedly implicated in bystander responses to ionizing radiation [41-46]. Therefore, we assayed C26GM cultures for the presence of functional intercellular channels using a novel, highly sensitive approach [47], based on a combination of patch clamp and voltage imaging with the membrane potential reporter Vf.2.1.Cl [48]. Our results (Figure 4a) indicate that cultured C26GM cells form functional syncytia since (i) electrical signals delivered to the patch-clamped cell invaded a number of other cells in the culture and (ii) electrical coupling was reversibly abrogated by carbenoxolone (CBX), a widely used non-specific inhibitor of connexin-made channels [49]. qPCR analysis for five different connexins expressed in various tumors [50-52] singled out Cx43 as the predominant isoform expressed by C26GM cells, whereas Cx40 and Cx26 provide minor contributions (Figure 4b).

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