Reduced exosomal L-Plastin is responsible for radiation-induced bystander effect

Abstract

Radiation-induced bystander effects (RIBE) are discussed as relevant processes during radiotherapy. Irradiated cells are suggested to release growth-inhibitory/DNA-damaging factors transported to non-irradiated cells. However, the molecular nature of this phenomenon has not yet been resolved. We aimed at identifying the growth-inhibitory factor(s) transmitted to non-irradiated cells. RIBE-competent PC3 cells were used to produce conditioned medium (CM) after exposure to ionizing radiation. Indicator cells were incubated with CM and clonogenic survival as well as cell proliferation were determined as endpoints. A549 indicator cells exhibited a bystander effect upon incubation with CM from irradiated PC3 cells. This bystander effect was not due to DNA-damaging factors, but a radiation-triggered reduction of mitogenic/clonogenic activity present in CM. Several tumor cells, but not normal fibroblasts secrete this factor, whose release is reduced by irradiation. We identified L-Plastin to be responsible for the mitogenic/clonogenic activity. Removal of L-Plastin from CM by immunoprecipitation or siRNA-mediated knockdown of L-Plastin expression resulted in loss or reduction of mitogenic/clonogenic activity transmitted via CM, respectively. Exosome-transported L-Plastin was constitutively Ser5-phosphorylated, indicative of its bioactive conformation. In summary, we observed production and exosomal secretion of L-Plastin by cancer cells. Via exosome-transmitted L-Plastin, tumors induce clonogenic and mitogenic activity in cancer and normal cells of the tumor microenvironment. Irradiation inhibits L-Plastin production targeting both cancer cells and the tumor niche and may explain the high impact of radiotherapy in tumor control.

Keywords: Bystander effect; Exosomes; L-Plastin; RIBE.