Human thyroid FTC-133 cell line was obtained from European Collection of Cell Cultures (ECACC, Salisbury, UK). It derives from a lymph node metastasis of a follicular thyroid carcinoma and carries p53R273H mutation and EGFR overexpression. FTC-133 and WRO (wtp53) (ECACC) cancer cell lines were routinely maintained in DMEM-Ham's-F12 (Life Technology-Invitrogen), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin (Life Technology-Invitrogen), and 2 mmol/l L-glutamine in a humidified atmosphere with 5% CO₂ and 95% air at 37°C.

For treatments the following reagents were used: Zn(II)-curc (29,30) dissolved in DMSO was added to the culture medium to a final concentration ranging between 40 and 120 μM; chemotherapeutic agent adriamycin (ADR) (Sigma), dissolved in PBS was added to the culture medium to a final concentration ranging between 0.2 and 2 μg/ml and cisplatin (Teva Pharma-Italia, Italy) was added to the culture medium to a final concentration ranging between 0.5 and 5 μg/ml; PRIMA-1 (p53 reactivation and induction of massive apoptosis-1) (Sigma) was added to the culture medium to a final concentration 10 μM for 24 h. PBS and DMSO solvents were used as control.

Exponentially proliferating cells were plated at subconfluence in triplicate in 60-mm Petri dishes and 24 h later treated with Zn(II)-curc alone or combination with chemotherapeutic agents for 24 h. Both floating and adherent cells were collected and cell viability was determined by trypan blue exclusion by direct counting with a hemocytometer, as reported (31). The percentage of cell viability, as blue/total cells, was assayed by scoring 200 cells per well three times.

Total cell extracts were prepared by incubation at 4°C for 30 min in lysis buffer [50 mmol/l Tris-HCl, pH 7.5, 150 mmol/l NaCl, 150 mmol/l KCl, 1 mmol/l dithiothreitol, 5 mmol/l EDTA, pH 8.0, 1% Nonidet P-40] plus a mix of protease and phosphatase inhibitors (Roche Diagnostic). Cell lysates were spun at 15,000 g for 15 min to remove debris and collect the supernatant (total cell extracts). Protein concentration was then determined using BCA Protein Assay kit (Bio-Rad). Equal amount of total cell extracts were resolved according to molecular weight on 10–18% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinylidene difluoride membrane (PVDF, Millipore). Unspecific binding sites were blocked by incubating membranes for 1 h in 0.05% Tween-20 (v/v in TBS) supplemented with 5% non-fat powdered milk or bovine serum albumin, followed by overnight incubation with the following primary antibodies: LC3B (Sigma-Aldrich), rabbit polyclonal anti-p53 (FL393), rabbit polyclonal anti-p21 and anti-p62 (Santa Cruz Biotechnology, Dallas, TX, USA), (p)-Akt (Ser473), tot-Akt (Cell Signaling Technologies, Danvers, MA, USA), polyclonal anti-EGFR antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and purified mouse anti-phospho-histone H2AX (Ser139, γH2AX) (Millipore, clone JBW301). Equal lane loading was monitored by probing membranes with antibodies specific for β-actin (Calbiochem, San Diego, CA, USA) or tubulin (Immunological Sciences, Rome, Italy). Primary antibodies were detected with appropriate horseradish peroxidase-labeled secondary antibodies (Bio-Rad, Hercules, CA, USA). Enzymatic signals were visualized using chemiluminescence (ECL Detection system, GE Healthcare, Milan, Italy).

Cells were harvested in TRIzol reagent (Invitrogen) and total RNA was isolated following the manufacturer's instructions. The first strand cDNA was synthesized from 2 μg of total RNA with MuLV reverse transcriptase kit (Applied Biosystems, Foster City, CA, USA). Semi-quantitative RT-PCR was performed essentially as previously described (20). cDNA was synthesized from 2 μg of total RNA with MuLV reverse transcriptase kit (Applied Biosystems). Semi-quantitative reverse-transcribed (RT)-PCR was carried out by using Hot-Master Taq polymerase (Eppendorf S.r.l., Milan, Italy) with 2 μl cDNA reaction and gene-specific oligonucleotides under conditions of linear amplification. PCR products were resolved on 2% agarose gel and visualized with ethidium bromide staining using UV light. The 28S mRNA sequence was used as control for efficiency of RNA extraction and transcription. Densitometric analysis was applied to quantify mRNA levels compared to control gene expression.

ChIP analysis was carried out essentially as previously described (20). Briefly, cells were crosslinked with 1% formaldehyde for 10 min at room temperature and then inactivated by the addition of 125 mM glycine. Chromatin extracts containing DNA fragments with an average size of 500 bp were incubated overnight at 4°C with milk, shaking using polyclonal anti-p53 antibody (FL393, Santa Cruz Biotechnology). Before use, protein G (Pierce) was blocked with 1 μg/μl sheared herring sperm DNA and 1 μg/μl BSA for 3 h at 4°C and then incubated with chromatin and antibody for 2 h at 4°C. PCR was performed with Hot-Master Taq (Eppendorf) using 2 μl of immunoprecipitated DNA and promoter-specific primers spanning p53 binding sites. Immunoprecipitation with non-specific immunoglobulins (IgG, Santa Cruz Biotechnology) was performed as negative controls. PCR products were resolved on 2% agarose gel and visualized by ethidium bromide staining using UV light.

Each experiment, unless otherwise specified, was performed at least three times, and data are presented as the mean ± SD. Statistical significance was determined using Student's t-test and a P-value of ≤0.05 was considered statistically significant.

We previously reported that Zn(II)-curc induces mutp53 degradation through autophagy in breast cancer cells (28). As defects in autophagy may be found in cancer, we evaluated whether Zn-curc could induce autophagy in FTC-133 thyroid cancer cells. We evaluated the expression of microtubule-associated protein light chain 3 (LC3) protein that, after conversion from LC3-I to its autophagosome membrane-associated lipidated form LC3-II, is considered a cellular readout of autophagy, and the expression levels of p62, a bona fide autophagic substrate (32). As shown in Fig. 1, Zn(II)-curc increased LC3-II expression and reduced p62 levels; parallel to induction of autophagy, Zn(II)-curc triggered mutp53 downregulation in FTC-133 cells (Fig. 1). The use of chemical inhibitors of autophagic/lysosomal degradation cloroquine (CQ) (32) prevented Zn(II)-curc-induced mutp53 degradation (Fig. 1). These findings indicate that Zn(II)-curc induced mutp53 degradation in FTC-133 cells, carrying p52R273H mutation, likely through autophagy.

We then evaluated whether Zn(II)-curc was promoting reactivation of wtp53 function in FTC-133 cells. We first performed p53/DNA binding by ChIP analyses and found that Zn(II)-curc treatment indeed restored the wtp53 binding to several specific target promoters, including Puma, p53AIP1, MDM2, and p21, while abolished the p53 binding to the mutant p53 target promoter MDR1 (multi-drug resistance 1) (Fig. 2A). As a consequence, the expression of the canonical wtp53 target genes was evaluated by reverse transcription polymerase chain reaction (RT-PCR) analyses. FTC-133 cells were exposed to increasing doses of Zn-curc (40, 80 and 120 μM for 24 h) that induced expression of the typical p53 target genes already at the lowest dose used, while reduced the expression of mutp53 target MDR1 expression starting from 80 μM dose (Fig. 2B). These findings indicate that Zn(II)-curc produced an imbalance between misfolded/folded p53 proteins in FTC-133 cells that favoured wild-type over mutant p53 functions.

PRIMA-1 is a low molecular weight compound with antitumor activity that has been shown to be more effective in inducing apoptosis in mutp53 cells than in wtp53 cells (33). We then compared the p53 reactivating effect of Zn(II)-curc with that of PRIMA-1 in both FTC-133 (mutp53) and WRO (wtp53) cells. In vivo transcription of endogenous p53 target genes was examined by employing RT-PCR analysis. The results show that the induction of wtp53 target genes such as Bax, Noxa, Puma and downregulation of the mutp53 target gene MDR1 was comparably and efficiently achieved by both treatments, that is, Zn(II)-curc and PRIMA-1 (Fig. 3A); of note, Zn(II)-curc induced also the wtp53 target, cell cycle-related p21 gene, unlike PRIMA-1 (Fig. 3A). This latter result is in agreement with PRIMA-1 pro-apoptotic specific function (33). Interestingly, Zn(II)-curc, differently from PRIMA-1, induced p53 target genes in WRO cells, carrying wtp53 (Fig. 3B), likely due to the DNA intercalating ability of Zn(II)-curc compound sensing the DNA damage response (29). Thus, Zn-curc induced H2AX phosphorylation (γH2AX) (Fig. 3C) that is a marker to detect the genotoxic effect of different anti-cancer agents (34).

P53H273 mutation has been shown to increase epidermal growth factor receptor (EGFR) levels as well as EGFR/PI3K/Akt signaling pathway, facilitating cell proliferation, tumor growth and migration (35). EGFR overexpression is described in several thyroid carcinomas, including follicular thyroid carcinomas (36), and recent evidence implicates EGFR overexpression in advanced thyroid carcinoma progression (37). Therefore, we evaluated the effect of Zn(II)-curc on EGFR and Akt expression levels in FTC-133 cells. Western blot analyses show that the strong expression of EGFR was markedly reduced by Zn(II)-curc (Fig. 4A); similarly, Akt phosphorylation was noticeably inhibited by Zn(II)-curc treatment (Fig. 4B). Moreover, p53 mutants have been shown to regulate E-cadherin expression (38). Downregulation of the E-cadherin cell-cell adhesion molecule is a key event for epithelial to mesenchymal transition (EMT) in tumor progression and it is associated with the development of invasive carcinoma, metastatic dissemination, and poor clinical prognosis (39). We found that Zn(II)-curc induced re-expression of E-cadherin mRNA levels in FTC-133 cells, that strongly correlated with downregulation of N-cadherin expression, a mesenchymal marker (Fig. 4C), as evidenced by densitometric analysis (Fig. 4C, lower panel) indicative of a reversion of EMT phenotype. Altogether, these findings indicate that Zn(II)-curc could modify prosurvival pathways and counteract mutp53-regulated pathways responsible of GOF.

In order to study the biological effect of Zn(II)-curc we treated FTC-133 and WRO cells with Zn(II)-curc alone or combination with cisplatin (cis) or adriamycin (ADR) and assessed viability using trypan blue staining. We found that the weak antitumor effect achieved by two different doses (0.2 and 2 μg/ml) of ADR in FTC-133 cells was significantly increased by Zn(II)-curc co-treatment (Fig. 5A). Similarly, cell death induced by both low (0.5 μg/ml) or high (5 μg/ml) dose of cisplatin was increased by Zn(II)-curc co-treatment, although FTC-133 resulted more sensitive to high dose of cisplatin (5 μg/ml) (Fig. 5A); in agreement with the activation of wtp53 target genes, seen in Fig. 3B, Zn(II)-curc increased the cytotoxic effect of cisplatin and ADR also in this wtp53-carrying cell line (Fig. 5B). In comparison, PRIMA-1 increased chemosensitivity of mutp53 FTC133 cells, while did not affect the response to drugs of wtp53 WRO cells (Fig. 5C). Altogether, these results show that Zn(II)-curc enhanced the cytotoxic effect of chemotherapeutic drugs in both mutant and wild-type-carrying cancer cells, unlike PRIMA-1 that was effective only in mutp53 cells.