A 779

Human adrenocortical cancer NCI-H295R cells (ATCC/LGC Standards S.r.L., Milan, Italy) were grown in RPMI medium supplemented with 10% fetal bovine serum (FBS), 1% glutamine and 1% antibiotic/antifungal mixture. They were seeded in 12-well plates at 2 × 10⁵ cells for well, grown to sub-confluence (80%) and starved for 24 h with RPMI medium supplemented with 0.5% FBS, 1% glutamine and 1% antibiotic/antimycotic before treatment. The cells were treated with DIZE at concentrations ranging from (10^–7 to 10^–4 mol/l), alone or in presence of (10^–7 mol/l) Ang II (Sigma-Aldrich). NCI-H295R cells were also treated with increasing concentrations of Ang-(1–7), from 10^–8 to 10^–4 mol/l (Sigma-Aldrich), alone or in addition to 10^–7 mol/l Ang II.
The selective MasR antagonist A779 (Abcam, Cambridge, UK) and the selective AT1R antagonist irbesartan (Sigma-Aldrich) were added to fresh media 30 min before treatment with Ang-(1–7).
To investigate the potential cytotoxicity of DIZE, cells were stimulated for 12 h with 10⁻⁵ and 10⁻⁴ mol/l DIZE, and counted after trypsin treatment using the trypan blue assay.

Animals received PNA6, a lactosyl Ang-(1-7) analog, or vehicle (saline) in the absence or presence of the MasR1 antagonist A-779 (Abcam, Cambridge, MA, USA). All injections were made by the intraperitoneal (i.p.) route at a dose of 1 mg/kg (10 mL/kg). In the antagonist studies, A-779 (1 mg/kg, i.p.) was administered 30 min before PNA6 administration. Therefore, only one PNA6 injection (1 mg/kg i.p.) or vehicle (saline) was given on day seven of post-cancer cells femur inoculation for the acute inflammatory study. For the chronic study, administration of PNA6 or vehicle (saline) was started on day seven post-cancer cell femur inoculation and continued with once-daily injections of PNA6 (1 mg/kg, i.p) or vehicle for seven consecutive days. Dosing was performed regularly at 9:00 am. Lastly, for the CIPN study, oxaliplatin (4 mg/kg i.p.) (Tocris Bioscience a Bio-Techne Brand, 61825-94-4, catalog No.: 2623) was prepared daily in a vehicle of sterile 5% dextrose solution distilled water. Systemic administration of PNA6 (1 mg/kg, i.p) or vehicle was started on day one and continued with once-daily injection for 14 consecutive days.