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

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 promoted 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.

The Role of Caspase-12 in Retinal Bystander Cell Death and Innate Immune Responses against MCMV Retinitis

(1) Background: caspase-12 is activated during cytomegalovirus retinitis, although its role is presently unclear. (2) Methods: caspase-12−/− (KO) or caspase-12+/+ (WT) mice were immunosup eyes were analyzed by plaque assay, TUNEL assay, immunohistochemical staining, western blotting, and real-time PCR. (3) Results: increased retinitis and a more extensive virus spread were detected in the retina of infected eyes of KO mice compared to WT mice at day 14 p.i. Compared to MCMV injected WT eyes, mRNA levels of interferons α, β and γ were significantly reduced in the neural retina of MCMV-infected KO eyes at day 14 p.i. Although similar numbers of MCMV infected cells, similar virus titers and similar numbers of TUNEL-staining cells were detected in injected eyes of both KO and WT mice at days 7 and 10 p.i., significantly lower amounts of cleaved caspase-3 and p53 protein were detected in infected eyes of KO mice at both time points. (4) Conclusions: caspase-12 contributes to caspase-3-dependent and independent retinal bystander cell death during MCMV retinitis and may also play an important role in innate immunity against virus infection of the retina.

La Crosse Virus Infection of Human Keratinocytes Leads to Interferon-Dependent Apoptosis of Bystander Non-Infected Cells In Vitro

Resident cells in the skin serve as the first innate line of defense against insect-borne pathogens, but the role of these cell types in promoting or limiting arbovirus replication is not completely understood. Here, we have examined the outcome of infection of cultured human keratinocyte cells with La Crosse virus (LACV), using a spontaneously transformed cell line, HaCaT. In single cycle infections, keratinocyte HaCaT cells supported rapid and high level LACV replication, resulting in high virus yields and extensive caspase-dependent cell death. By contrast, multi-cycle LACV replication in HaCaT cells was restricted by an antiviral response elicited by the production of both IFN-β and IFN-λ. During low multiplicity LACV infections, HaCaT cell death was seen in non-infected bystander cells. Media from LACV-infected cells induced caspase-dependent killing of naïve non-infected HaCaT cells, and this bystander cell death was relieved by IFN-β neutralizing antibodies or by an inhibitor of JAK-STAT signaling. Naïve HaCaT cells showed dose-dependent killing by treatment with exogenous IFN-β but not IFN-λ. Our data suggest a model whereby keratinocytes produce IFNs which limit virus spread through both antiviral signaling and by induction of bystander cell death of potential new target cells for infection.

Photosensitizer Activation Drives Apoptosis by Interorganellar Ca2+ Transfer and Superoxide Production in Bystander Cancer Cells

In cells, photosensitizer (PS) activation by visible light irradiation triggers reactive oxygen species (ROS) formation, followed by a cascade of cellular responses involving calcium (Ca2+) and other second messengers, resulting in cell demise. Cytotoxic effects spread to nearby cells not exposed to light by poorly characterized so-called “bystander effects”. To elucidate the mechanisms involved in bystander cell death, we used both genetically encoded biosensors and fluorescent dyes. In particular, we monitored the kinetics of interorganellar Ca2+ transfer and the production of mitochondrial superoxide anion (O2−∙) and hydrogen peroxide (H2O2) in irradiated and bystander B16-F10 mouse melanoma cancer cells. We determined that focal PS photoactivation in a single cell triggers Ca2+ release from the endoplasmic reticulum (ER) also in the surrounding nonexposed cells, paralleled by mitochondrial Ca2+ uptake. Efficient Ca2+ efflux from the ER was required to promote mitochondrial O2−∙ production in these bystander cells. Our results support a key role for ER–mitochondria communication in the induction of ROS-mediated apoptosis in both direct and indirect photodynamical cancer cell killing.