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

Abstract

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.

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

“…On average, lowering the [Ca 2+ ] ex in the DSC with EGTA increased ~four-fold the variation of [Ca 2+ ] cyt in bystander cells (Figure 1B,C). The simplest explanation for this seemingly paradoxical effect is that the ER was the main source of the observed [Ca 2+ ] cyt increments, which is consistent with previous results using AlPcCl as PS [22].…”

“…For fPDT, a single cell at the surface of the tumor was irradiated with light from a fiber-coupled 671-nm diode-pumped solid-state laser (Shanghai Dream Lasers Technology, Shanghai, China), as described in [22]. Briefly, the recollimated laser beam was injected into the microscope optical path just above the 60× water immersion objective (Nikon Fluor 60× Water, NA = 1.0, WD = 2 mm, Nikon, Tokyo, Japan) and focused into a 10-µm ∅ spot.…”

“…where F 0 denotes pre-stimulus value, were carried out as previously described [22]. Time integrals of ∆F/F 0 traces were computed with the Matlab function trapz().…”

“…Our published results with aluminum phthalocyanine chloride (AlPcCl) in tumorigenic murine cell lines indicate that photoactivation of this PS triggers apoptosis in bystander tumor cells via propagation of intercellular Ca 2+ waves that proceed from irradiated to bystander cells. AlPcCl activation also promotes NO production, a gaseous molecule that diffuses freely across plasma membranes and can enter bystander cells, in which NO concentration is further increased by a mechanism compatible with Ca 2+ -dependent enzymatic production [20][21][22].…”

Connexins in melanoma: Potential role of Cx46 in its aggressiveness

“…Similar results were obtained using regular 5-FU anticancer therapy in combination with eicosapentaenoic acids (EPA) that upregulate Cx43, enhancing the drug cytotoxicity through the activation of mitogen-activated protein kinase (MAPK) signaling pathways (Yang et al, 2019). In addition, the bystander effect is the basis of gene suicide therapy success, where dying cells share toxic substrates via GJCs to neighbor cells, promoting massive cell death (Nardin et al, 2019). Accordingly, the treatment of B16 melanoma cells with tanshinone (Tan IIA) or dioscin (chemical substances extracted from roots), induces the upregulation of Cx43 and the consequent formation of functional Cx43-GJ-like structures that allow the bystander effect of gene suicide therapy and ionizing radiation therapies (Ohshima et al, 2012;Xiao et al, 2013 andXiao et al, 2017).…”