Human breast cancer is a major cause of global morbidity and mortality in women and it is a process that involves numerous molecular and cellular alterations attributed to environmental substances and agents such as hormones. Bisphosphonates, such as pamidronate, are potent antiresorptive drugs used to the treatment of metabolic bone disease, exerting anti-proliferative, anti-migratory and apoptotic effects. The aim of this study was to evaluate gene and protein expression involved in these processes. An in vitro model was developed with the MCF-10F immortalized breast epithelial cell line exposed to low radiation doses of high LET (linear energy transfer) α-particles (150 keV/μm) and cultured in the presence of 17β-estradiol (estrogen). This model consisted of the following cell lines: i) MCF-10F, normal; ii) Alpha3, non-malignant; iii) Alpha5, pre-tumorigenic, and iv) Tumor2, derived from Alpha5 injected into the nude mice. Our previous results have shown that Alpha5 and Tumor2 increased cell proliferation, anchorage independency, invasive capabilities and tumor formation in nude mice in comparison to control. Expression of the gene (RT-qPCR) and protein (western blotting, flow cytometry) was measured. The results indicated that pamidronate decreased invasion, migration and Rho-A, c-Ha-ras, p53, Serpin-1, Caveolin-1, Bcl-xL and NFκB gene and protein expression. Thus, it seems that pamidronate may impinge upon cellular proliferation, invasion, metastasis and apoptosis and it may exert antitumor activity in breast cancer cells transformed by low doses of α-particles and estrogen in vitro.