Effect of zoledronic acid and leuprorelin acetate on proliferative and migratory properties of prostate cancer cells in vitro.

B Mognetti1, G La Montagna1, MG Perrelli1, C Cracco0,2, C Penna1. 1Università degli Studi di Torino, Dpt of Clinical and Biological Science, 10043, Italy, 2Ospedale Cottolengo di Torino, S.C Urologia, 10152, Italy

Prostate cancer is the most frequent genitourinary cancer. Current treatments consist in androgen ablation, mostly obtained with GnRH analogues. Despite improvements in therapies, however it is still the third specific death cause, mostly because of metastasis. Bone is a preferential site for prostate cancer metastasis and bisphosphonates therapy is often associated since it increases bone mass density and prevent osteopenia and osteoporosis. Several in vitro studies have shown that bisphosphonates exert direct cytostatic and proapoptotic effects on a variety of human tumor cell lines. Since prostate cancer patients are often treated simultaneously with zoledronic acid (bisphosphonate-ZA) and leuprorelin acetate (LHRH – Luteinizing hormone releasing hormone – analogous, LA), and since drug interactions are quite common at various levels, the aim of this work has been to study, in vitro, the effects of the single drugs and of their association on two different human cell lines derived from prostate cancer. Particularly, we focused on some crucial aspects that drugs might play in tumor evolution and metastatization, such as cytotoxicity, migration and interactions with bone cells. Experiments were performed on PC-3 and DU-145 human prostate cancer cell lines, statistical significance was calculated by one- or two-way analysis of variance. ZA cytotoxicity has been confirmed on both cell lines after 48 hours incubation (p=0.006 and 0.020 for PC-3 and DU-145 respectively) at the 10 µM concentration, while LA cytotoxicity appears only after 72hrs contact, at much higher concentrations. Coincubation (ZA+LA) does not increase ZA cytotoxicity. Both ZA 5 µM and (even more significantly) LA 100 µM decreased 2D (wound healing) and 3D (transwell migration test) migration rate of PC-3 and DU-145 cells. pErk/Erk ratio is similarly increased by LA and ZA; pAkt/Akt ratio is decreased by LA and, in less extent by ZA, in agreement with the respective inhibition migration ratios. Cells underwent migration test in mesenchymal stem cells conditioned medium (MSC), which significantly increases the rate of migration (PC-3: + 291.73% ± 11.85, p = 0.001; DU-145: (+ 76.42% ± 4.37, p = 0.006) and cell dimensions (PC-3: + 41.77%; DU-145: + 97.95%). Addition of both ZA and LA quenched the attractive effect of conditioned medium. Addition of the CXCL12 blocker AMD3100 greatly decreased the attractive effect of conditioned medium, suggesting the presence of CXCL12 in MSC conditioned medium and its important role for migration.

Our results suggest that drugs used in prostate cancer treatment have a direct toxic effect on cancer cells, mostly ZA. Those same drugs inhibit cellular migration even under attractive stimuli exerted by MSC, and this might contribute to explain their effect in limiting metastatization. CXCL12 is a good candidate to for the attractive effect exerted on prostate cancer cells by MSC. The main result is, however, the observation that the two cell lines considered have different sensibility to drugs and this is in line with the needs of a phenotype oriented therapy.