SOLUBLE FACTORS FROM RADIATION-INDUCED SENESCENT TUMOR CELLS REDUCES GROWTH OF NON-IRRADIATED TUMOR CELLS

SALVARREDI, L; AGUERO, H; BUSCEMA, V; CALLEGARI, E; CASTRO C; LOPEZ , L

MENDOZA

Congreso; LVIII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; 2023

SAIB

Radiation therapy is one of the most important options in treating cancer. Based on DNA damage, ionizing radiation (IR) activates different cell death programs such as apoptosis, autophagy, and senescence. The latter is characterized by an irreversible cell cycle arrest and the secretion of a protein profile known as the senescence-associated secretory phenotype (SASP). SASP can inducemultiple effects on neighboring cells not exposed to radiation (naïve cells), a phenomenon that in radiotherapy is part of the so-called bystander effects (BE). The aim of this work was to evaluate whether SASP from radiation-induced senescent cells produces by naive tumor cells. Murine melanoma B16F0 cell line cultures were exposed to 0 or 10 Gy doses of IR (NI-B16F0 and IRB16F0cells) and cell growth and clonogenic capacity were reduced. To explore the mechanism involved, apoptosis and senescence were analyzed. Apoptosis was not observed but cells became senescent 3 days after radiation. To explore the biological activity of SASP from B16F0 senescent cells, conditioned media from IR or NI cells were harvested on day 3 after radiation (IR-CM and NICM).Naive B16F0 cells were incubated with NI/IR-CM and cell growth and migration were analyzed. IRCM reduced cell growth but not migration. To further explore if IR-CMs affected non-cancer cells, NIH 3T3 cell line was incubated with CMs. No significant changes in cell growth were observed. During the growth studies, it became apparent that B16F0 cells exposed to IRCM exhibited morphological features indicative of senescence, which led us to histochemically stain for the senescence marker SA-β-gal. As predicted, B16F0 cells treated with IR-CM exhibited pronounced SA-β-gal positivity. The protein profile present in the CMs was characterized. Proteins from IR-CMs and NI-CMs were separated in large polyacrylamide gels. IR-CMs presented a greater number of bands compared to NI-CMs indicating an increased protein diversity. Furthermore, aliquots from the same CMswere analyzed by means of two-dimensional nano-liquid chromatography-mass spectrometry (2DnanoLC-MS/MS) with the aim of identifying specific proteins. The in silico analysis confirmed that IR-CMs presented higher protein diversity than NI-CMs. Using bioinformatics tools, biological processes and pathways of protein profiles contained in CMs were analyzed. IR-CMs expressed more proteins related to the positive regulation of cellular senescence and fewer proteins related to the positive regulation of cell growth and cell migration. In addition, IR-CMs were enriched in proteins related to oxidative stress-induced senescence. In conclusion, cells undergoing radiation-induced senescence reduce the growth of not exposed tumor cells by secretion of soluble factors associated with senescence induction and maintenance. The identification of these factors might help to identify new targets towards manipulating them for therapeutic benefit