Moreover, Zn-curc localized inside glioblastoma tissues suggesting its ability to cross the blood-tumor barrier. Materials

and methods Ethics statement All animals were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies, and in accordance with the Italian and European legislation. All work was performed in accordance with the guidelines of the National Cancer Institute Regina Elena, where there is currently no active Ethical Committee for animal research, MGCD0103 clinical trial and has been filed with the Veterinary Service Unit and the Italian Ministry of Health, in accordance with the Italian and European legislation. Cell culture and treatments The human colon cancer RKO (wtp53), glioblastoma U373MG (expressing R273H p53 mutation) and T98G (expressing M237I p53 mutation) cell lines were maintained in RPMI-1640 (Life Technology-Invitrogen), while human SKBR3 (expressing R175H p53 mutation), MD-MBA231 (expressing p53 mutation R280K) breast cancer cell lines and human fibroblasts (HF) (kindly provided by S. Soddu, Regina Elena National Cancer Institute, Rome, Italy) were maintained in DMEM (Life Technology-Invitrogen), all supplemented with 10% heat-inactivated fetal bovine serum plus glutamine and antibiotics. The following reagents were used: a heteroleptic pentacoordinated (bpy-9)Zn(curc, Cl) complex containing a

4,4′-disubstituted-2,2′-bipyridine as main ligand and curcumin (curc) and chloride (Cl) as ancillary ligands [13] was 17-DMAG (Alvespimycin) HCl dissolved in DMSO and used at the LY3023414 purchase indicated concentrations; curcumin was prepared as previously reported [17]; pifithryn-α (PFT-α) (ENZO Life Sciences, Lausen Switzerland) was dissolved in DMSO and used at 30 μM; adryamycin (ADR) was used at 2 μg/ml and ZnCl2 was used at 100 μM. Viability and colony assays Subconfluent cells were plated in triplicate in 60 mm Petri BI 2536 concentration dishes and 24 h later treated with Zn-curc complex (20-50-100 μM) for 24 and 48 h. Both floating and adherent cells were collected and cell viability was determined by Trypan blue exclusion by direct counting with a haemocytometer, as reported. The percentage

of cell viability, as blue/total cells, was assayed by scoring 200 cells per well three times. For long-term cell survival, subconfluent cells were plated in 60 mm Petri dishes and 24 h later treated with Zn-curc complex (20-50-100 μM). Twenty-four hours later, plates were washed with PBS and fresh medium was added. Death-resistant colonies were stained with crystal violet 14 days later. Cell death/PI staining Cell death was detected by cytofluorimetric analysis of propidium iodide (PI)-stained cells staining. Briefly, cells floating were collected by centrifugation and pooled with adherent cells recovered from the plates, fixed in 80% ethanol and stained in a PBS solution containing PI (62.5 mg/mL; Sigma-Aldrich), and RNase A (1.125 mg/mL; Sigma-Aldrich).