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. 2013 Oct 7;32(1):72.
doi: 10.1186/1756-9966-32-72.

A fluorescent curcumin-based Zn(II)-complex reactivates mutant (R175H and R273H) p53 in cancer cells

Alessia Garufi, Daniela Trisciuoglio, Manuela Porru, Carlo Leonetti, Antonella Stoppacciaro, Valerio D'Orazi, Maria Avantaggiati, Alessandra Crispini, Daniela Pucci, Gabriella D'Orazi

A fluorescent curcumin-based Zn(II)-complex reactivates mutant (R175H and R273H) p53 in cancer cells

Alessia Garufi et al. J Exp Clin Cancer Res. .

Abstract

Background: Mutations of the p53 oncosuppressor gene are amongst the most frequent aberration seen in human cancer. Some mutant (mt) p53 proteins are prone to loss of Zn(II) ion that is bound to the wild-type (wt) core, promoting protein aggregation and therefore unfolding. Misfolded p53 protein conformation impairs wtp53-DNA binding and transactivation activities, favouring tumor growth and resistance to antitumor therapies. Screening studies, devoted to identify small molecules that reactivate mtp53, represent therefore an attractive anti-cancer therapeutic strategy. Here we tested a novel fluorescent curcumin-based Zn(II)-complex (Zn-curc) to evaluate its effect on mtp53 reactivation in cancer cells.

Methods: P53 protein conformation was examined after Zn-curc treatment by immunoprecipitation and immunofluorescence assays, using conformation-specific antibodies. The mtp53 reactivation was evaluated by chromatin-immunoprecipitation (ChIP) and semi-quantitative RT-PCR analyses of wild-type p53 _target genes. The intratumoral Zn-curc localization was evaluated by immunofluorescence analysis of glioblastoma tissues of an ortothopic mice model.

Results: The Zn-curc complex induced conformational change in p53-R175H and -R273H mutant proteins, two of the most common p53 mutations. Zn-curc treatment restored wtp53-DNA binding and transactivation functions and induced apoptotic cell death. In vivo studies showed that the Zn-curc complex reached glioblastoma tissues of an ortothopic mice model, highlighting its ability to crossed the blood-tumor barrier.

Conclusions: Our results demonstrate that Zn-curc complex may reactivate specific mtp53 proteins and that may cross the blood-tumor barrier, becoming a promising compound for the development of drugs to halt tumor growth.

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Figures

Figure 1
Figure 1
Zn-curc impairs survival of mutant p53-carrying cells. (A) Tumor cells (4 x 104) were plated in 60 mm dish and 24 h later treated with increased amount of Zn-curc (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. (B) Death-resistant colonies as in (A) were counted and plotted as percentage ± SD of two independent experiments performed in duplicate. (C) Cells (3 x 105) were plated at subconfluence in 60 mm dish and the day after treated with Zn-curc for 24 and 48 h. Cell viability was measured by trypan blue exclusion assay and expressed as percentage ± SD of two independent experiments. (D) Cytofluorimetric analysis of the SubG1 peak evaluated by Propidium Iodide (PI) staining (upper panel) and microscopical analysis of SKBR3 cells, mock-treated or treated with Zn-curc (100 μM) for 24 h (lower panel). Percentage of apoptotic cells is shown ± SD of two independent experiments. (E) SKBR3 and U373 cells were treated with Zn-curc (100 μM) for 24 h. Equal amount of total cell extracts were subjected to immunoblot with anti-PARP (cleaved form, 87 Kd) or anti-β-actin antibodies. (F) RKO cells were treated with Zn-curc (100 μM), ZnCl2 (100 μM) or adryamicin (ADR, 2 μg/ml) for 24 h. Equal amount of total cell extracts were subjected to immunoblot with anti-γH2AX (phopho-Ser139) or anti-β-actin antibodies.
Figure 2
Figure 2
Zn-curc restores wild-type p53-DNA binding and transactivating activities. (A) SKBR3 and U373 cells (6x106) were plated in 150 mm dish and the day after treated with Zn-curc (100 μM) for 16 h before assayed for chromatin immunoprecipitation analysis (ChIP) with anti-p53 or anti-p73 antibodies. PCR analyses were performed on the immunoprecipitated DNA samples using primers specific for wtp53 _target gene promoters (p21, Puma, p53AIP1, MDM2) or for mtp53 _target promoters (MDR1, cyclin B2). A sample representing linear amplification of the total chromatin (Input) was included as control. Additional controls included immunoprecipitation performed with non-specific immunogloblulins (No Ab). (B) Cells (3x105) were plated at subconfluence in 60 mm dish and the day after treated with Zn-curc for 24/48 h. p53 _target genes were detected by RT-PCR analysis. Gene expression was measured by densitometry and plotted as fold of mRNA expression over control (Mock), normalized to β-actin levels, ±SD. (C) SKBR3 and U373 cells were plated at subconfluence in 60 mm dish and the day after treated with Zn-curc (100 μM) for 24 h, with or without p53 inhibitor pifithrin-α (PFT-α) (30 μM). p53 _target genes were dtected by RT-PCR analysis. β-actin was used as control. (D) Gene expression as in (C), was measured by densitometry and plotted as fold of mRNA expression over control (Mock), normalized to β-actin levels, ±SD. (E) SKBR3 and U373 cells were treated with Zn-curc (100 μM) for the indicated hours and total cell extracts were subjected to immunoblot analysis. (F) U373 cells were plated at subconfluence in 60 mm dish and the day after treated with curcumin (Curc) (50, 100 μM) for 24 h. Zn-curc (100 μM for 24 h) was used as control of p53 activation. p53 _target genes were detected by RT-PCR. β-actin was used as control.
Figure 3
Figure 3
Zn-curc induces a wild-type-like conformational change in mutant p53 proteins. (A) Immunofluorescence of SKBR3 (H175) and U373 (H273) cells using p53-conformation-specific antibodies (PAB1620 for wt, folded conformation and PAB240 for mutant, unfolded conformation). Cells were treated with Zn-curc (100 μM) for 24 h before fixing and staining with antibodies. The RKO (wtp53) cell line is used as a control to show that the wtp53 conformation is not changed by Zn-curc treatment. Quantification of SKBR3 (B) or RKO (C) positive cells to PAB1620 and PAB240 antibodies before and after Zn-curc treatment, ±SD. (D) SKBR3 and U373 cells were treated with Zn-curc (100 μM) for 24 h. Total cell extracts were imunoprecipitated (IP) with conformation-specific antibodies (PAB1620 and PAB240) and then imunoblotted (WB) with anti-p53 (DO1) antibody. Input represents 1/10 of total cell extracts used for IP.
Figure 4
Figure 4
Zn-curc reactivates mtp53 in an orthotopic U373 glioblastoma model. (A) U373MG-LUC cells (2.5x105) were injected into the brain of athymic mice and left to growth for 6 days before treating animals with Zn-curc every day for 7 days. Mock- or Zn-curc-treated U373M-derived tumors were then harvested and analysed with a fluorescent microscope that showed as diffuse fluorescence only in Zn-curc-treated tumors. (B) Quantification of tumor cell fluorescence positivity in U373-derived tumors, untreated or Zn-curc-treated, ±SD. (C) Total mRNA was extracted from harvested U373-derived tumors, untreated or Zn-curc-treated, and p53 _target gene expression as well as VEGF, MDR1 and Bcl2 expression were assayed by PCR of reverse-transcribed cDNA. Gene expression was measured by densitometry and plotted as fold of mRNA expression over control (Mock), normalized to β-actin levels, ±SD.
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