AbstractRadio-induced apoptosis is mediated by the activation of tumor protein p53, Bax and caspases. The purpose of this study was to investigate the early activation of this pathway in men receiving in vivo irradiation immediately before radical prostatectomy for locally advanced prostate cancer. We also investigated cell proliferation index (Ki-67), proto-oncogene (p53) and anti-apoptotic protein (Bcl-2) levels as potential predictive factors. We selected a homogeneous sample of 20 patients with locally advanced prostate cancer and candidate to radical prostatectomy. To assess the apoptotic pathways, Bax, is studied through immunofluorescence assay, before and after 12 Gy single dose intraoperative radiotherapy (IORT) to the prostate, on bioptic samples and on surgical specimens. Moreover, before and after IORT, Bcl-2, p53, and Ki-67 were also detected through immunohistochemistry. A count of positive Bax spots for immunofluorescence was performed on tumor cells, prostatic intraepithelial neoplasia (PIN), and healthy tissue areas before and after IORT. We also analyzed Caspases 3 and 9 expressions after IORT. Before IORT, Bcl-2 mean value in neoplastic cells was 2.23% ± 1.95, mean Ki-67 in neoplastic area was 4.5% ± 3.8, and p53 was 22.5% ± 6.8. After IORT, Bcl-2 mean value in neoplastic cells was 8.85 ± 8.92%, Ki-67 in neoplastic area was 7.8 ± 6.09%, and p53 was 24.9 ± 26.4%. After the irradiation, healthy areas expressed significantly lower levels of Bax (2.81 ± 1.69%) with respect to neoplastic cells (p < 0.0001), while in PIN areas, Bax positive cells were significantly more present than in neoplastic areas (p = 0.0001). At statistical analysis, it was observed that cancer cells with Ki-67 ≥ 8% had a trend toward greater expression of Bax (p = 0.0641). We observed an increase of Bcl-2 expression after IORT in neoplastic areas (p = 0.0041). Biopsy specimens with p53 ≥ 18% and Ki-67 ≥ 8% had worse post-operative staging with extracapsular invasion (p = 0.04 for both parameters) and nodal positivity (p = 0.04 for p53 and p = 0.0001 at pathology for ki-67). No correlation between IORT and Caspases activation was noted. In conclusion, after 12 Gy IORT, Bax was overexpressed in tumor and PIN cells. Pre-operative Ki-67 and p53 definition could be used in future studies to predict patients with worse pathological stage, while Bcl-2 activation after IORT might be a predictive factor for loco-regional failure.