Reactivation of mutant p53 by capsaicin, the major constituent of peppers

Abstract

Background: Mutations in the p53 oncosuppressor gene are highly frequent in human cancers. These alterations are mainly point mutations in the DNA binding domain of p53 and disable p53 from transactivating target genes devoted to anticancer activity. Mutant p53 proteins are usually more stable than wild-type p53 and may not only impair wild-type p53 activity but also acquire pro-oncogenic functions. Therefore, targeting mutant p53 to clear the hyperstable proteins or change p53 conformation to reactivate wild-type p53 protein functions is a powerful anticancer strategy. Several small molecules have been tested for p53 reactivation in mutant p53-carrying cells while studies exploiting the effect of natural compounds are limited. Capsaicin (CPS) is the major constituent of peppers and show antitumor activity by targeting several molecular pathway, however, its effect on mutant p53 reactivation has not been assessed yet. In this study we aimed at investigating whether mutant p53 could be a new target of capsaicin-induced cell death and the underlying mechanisms.

Methods: p53 levels were analysed by western blot upon capsaicin treatment in the presence of the autophagy inhibitor chloroquine. The mutant p53 reactivation was evaluated by chromatin-immunoprecipitation (ChIP) assay and semi-quantitative RT-PCR analyses of wild-type p53 target genes. The specific wild-type p53 activation was determined by using the inhibitor of p53 transactivation function, pifithrin-α and siRNA for p53.

Results: Here, we show that capsaicin induced autophagy that was, at least in part, responsible of mutant p53 protein degradation. Abrogation of mutant p53 by capsaicin restored wild-type p53 activities over mutant p53 functions, contributing to cancer cell death. Similar effects were confirmed in cancer cells bearing tumor-associated p53 mutations and in H1299 (p53 null) with overexpressed p53R175H and p53R273H mutant proteins.

Conclusion: These findings demonstrate for the first time that capsaicin may reduce mutant p53 levels and reactivate wild-type p53 protein in mutant p53-carrying cells and the p53 reactivation contributes to capsaicin-induced cell death.

Keywords: Apoptosis; Autophagy; Capsaicin; Mutant p53; Natural compounds; p53; p53 reactivation.

Fig. 1

CPS induces cell death in mutp53-expressing cells in a p53-dependent manner. a U373 and SKBR3 cells were plated at subconfluence and the day after treated with CPS (100 and 200 μM). Twenty-four hours later, the percentage of dead cells was scored by trypan blue staining. Error bars show standard deviation. b H1299 cells were transiently transfected with pcDNA3-p53R175H (0.1 μg), pcDNA3-R273H (0.1 μg) and control pcDNA3 vectors and the day after transfection treated with CPS (200 μM) for 24 h. Cell death measurements were assayed by Tunel assay. The results are the mean of three independent experiments performed in triplicate ± SD. *p = 0.001. c U373 and SKBR3 cells were treated with CPS (200 μM) for 16 and 24 h. Equal amount of total cell extracts was analysed by western immunoblotting with anti-PARP antibody; the cleaved form is indicated by the arrow. Anti-β-actin was used as protein loading control

Fig. 2

p53 interference reduces the CPS-induced cell death in mutp53-expressing cancer cells. a U373 and SKBR3 were transfected with pSuper (si-ctr) or pSuper-p53 (si-p53) and 36 h after transfection equal amount of total cell extracts was analysed by western immunoblotting with anti-p53 antibody. Anti-β-actin was used as protein loading control. The percentage of p53 reduction was measured by densitometry and plotted in the right panel. b Control and interfered cells as in (a) were treated with CPS (200 μM). Twenty-four hours later, the percentage of dead cells was scored by trypan blue staining. Data are the mean ± S.D. of three independent experiments performed in triplicate. *P = 0.001

Fig. 3

CPS induces mutp53 protein degradation. a U373 and SKBR3 cells were treated with CPS (200 μM) and chloroquine (CHQ) (25 μM) for 24 h. Protein levels were measured with western blot using antibodies to LC3-II and p53. Anti-β-actin was used as protein loading control. In the right panel data are presented as mean ± S.D. *P = 0.001. b H1299 cells were transiently transfected with pcDNA3-p53R175H (0.1 μg), pcDNA3-R273H (0.1 μg) and control pcDNA3 vectors and the day after transfection treated with CPS (200 μM) for 24 h. Cell death measurements were assayed by Tunel assay. Equal amount of total cell extracts was analysed by western immunoblotting with anti-p53 and anti-p62 antibodies. Anti-β-actin was used as protein loading control. The percentage of p53 reduction was measured by densitometry and plotted in the right panel. *P = 0.001

Fig. 4

CPS restores wtp53 activities in mutp53-carrying cells. a U373 and SKBR3 were treated with CPS (200 μM) for 24 h. Western immunoblotting was performed on equal amount of total cell extracts to detect phospho-Histone H2A.X (γH2AX) levels. Anti-β-actin was used as protein loading control. b SKBR3 and U373 cells (6x10⁶) were plated in 150 mm dish and the day after treated with CPS (200 μM) for 16 h before being 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 promoter (Puma) or for mtp53 target promoter (MDR1). 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). SKBR3 and U373 cells were plated at subconfluence in 60 mm dish and the day after treated with CPS (200 μM) for 24 h, with or without autophagy inhibitor chloroquine (CHQ) (c) or p53 inhibitor pifithrin-α (PFT-α) (30 μM) (d). p53 target genes were detected by RT-PCR analysis. β-actin was used as control. Gene expression was measured by densitometry, normalized to β-actin levels, ±SD (right panels) and plotted as fold of mRNA expression over control (Mock)

Fig. 5

CPS increases drug-induced cell death in mutp53-carrying cells. SKBR3 and U373 cells were plated at subconfluence and the day after treated with CPS (100 μM) and, respectively, with ADR (1.5 μg/ml) and cisplatin (CDDP, 2.5 μg/ml). Twenty-four hours later, the percentage of dead cells was scored by trypan blue staining. Error bars show standard deviation. *P = 0.001