Connexin Hemichannel Activation by S-Nitrosoglutathione Synergizes Strongly with Photodynamic Therapy Potentiating Anti-Tumor Bystander Killing.

Chiara Nardin,Guang Yang,Yuanyuan Kuang,Chiara Peres,Marcello Raspa,Sabrina Putti,Anna Maria Salvatore,Fabio Mammano,Francesco Chiani,Ferdinando Scavizzi,Claudia Colussi,Mauricio A Retamal,Abraham Tettey-Matey,Tiziana Orsini,Flavia Mazzarda

doi:10.3390/cancers13205062

Abstract

Simple SummaryBystander effects depend on direct cell-cell communication and/or paracrine signaling mediated by the release of soluble factors into the extracellular environment and may greatly influence therapy outcome. Although the limited data available suggest a role for intercellular gap junction channels, far less is known about the role of connexin hemichannels. Here, we investigated bystander effects induced by photodynamic therapy in syngeneic murine melanoma models in vivo. We determined that (i) photoactivation of a photosensitizer triggered calcium-dependent cell death pathways in both irradiated and bystander tumor cells; (ii) hemichannel activity and adenosine triphosphate release were key factors for the induction of bystander cell death; and (iii) bystander cell killing and antitumor response elicited by photodynamic therapy were greatly enhanced by combination treatment with S-nitrosoglutathione, which promoted hemichannel opening in these experimental conditions. Therefore, these findings in a preclinical model have important translational potential.In this study, we used B16-F10 cells grown in the dorsal skinfold chamber (DSC) preparation that allowed us to gain optical access to the processes triggered by photodynamic therapy (PDT). Partial irradiation of a photosensitized melanoma triggered cell death in non-irradiated tumor cells. Multiphoton intravital microscopy with genetically encoded fluorescence indicators revealed that bystander cell death was mediated by paracrine signaling due to adenosine triphosphate (ATP) release from connexin (Cx) hemichannels (HCs). Intercellular calcium (Ca2+) waves propagated from irradiated to bystander cells promoting intracellular Ca2+ transfer from the endoplasmic reticulum (ER) to mitochondria and rapid activation of apoptotic pathways. Combination treatment with S-nitrosoglutathione (GSNO), an endogenous nitric oxide (NO) donor that biases HCs towards the open state, greatly potentiated anti-tumor bystander killing via enhanced Ca2+ signaling, leading to a significant reduction of post-irradiation tumor mass. Our results demonstrate that HCs can be exploited to dramatically increase cytotoxic bystander effects and reveal a previously unappreciated role for HCs in tumor eradication promoted by PDT.

Highlights

It has long been known that damage to cells directly hit by ionizing radiation can propagate to neighboring non-irradiated cells giving rise to the so-called radiation-induced bystander effect [1]
We allowed tumors to develop in the dorsal skinfold chamber (DSC) for 4 to 5 days, thereafter we focally stimulated a single cell in the PS-loaded tumor mass with laser light (671 nm) focused in a 10-μm ∅ spot, a procedure which we refer to as Focal PDT (fPDT), and tracked the ensuing intercellular Ca2+ waves by intravital multiphoton microscopy [27] (Figure 1; see Materials and Methods for details)
Since mobilization of internal Ca2+ stores seemed to be a key process in melanoma response to PS activation in vivo, and given that endoplasmic reticulum (ER)-mitochondria Ca2+ transfer is a key regulatory mechanism in Ca2+-dependent apoptosis and anti-cancer therapies [39,40,41,42], we investigated inter-organellar Ca2+ signaling in B16-F10 tumors grown in DSCs and expressing Ca2+-selective genetically encoded indicators targeted to subcellular compartments (Figure 4D) [43]

Summary

Introduction

It has long been known that damage to cells directly hit by ionizing radiation can propagate to neighboring non-irradiated cells giving rise to the so-called radiation-induced bystander effect [1]. Bystander effects depend on direct cell-cell communication and/or paracrine signaling mediated by the release of soluble factors into the extracellular environment and Cxs, a family of membrane proteins with four transmembrane helices, are involved in both type of processes [9]. Two HCs, each one from different cells in contact, may form an intercellular gap junction channel (IGJC) after docking head-to-head in the extracellular space and IGJCs have been implicated in bystander cell killing triggered by gene therapy for cancer [11], as well as ionizing radiation [12]. The limited data available suggest a role for IGJCs in PDT-induced bystander effects, very little is known about the contribution of HCs [13]

Methods

Results

Discussion

Conclusion