TP53, one of the most important oncosuppressors, is frequently mutated in cancer. Several p53 mutant proteins escape proteolytic degradation and are highly expressed in an aberrant conformation often acquiring pro-oncogenic activities that promote tumor progression and resistance to therapy. Therefore, it has been vastly proposed that reactivation of wild-type (wt) function(s) from mutant p53 (mutp53) may have therapeutic significance. We have previously reported that Zn(II) restores a folded conformation from mutp53 misfolding, rescuing wild-type (wt) p53/DNA-binding and transcription activities. However, whether Zn(II) affects mutp53 stability has never been investigated. Here we show that a novel Zn(II) compound induced mutp53 (R175H) protein degradation through autophagy, the proteolytic machinery specifically devoted to clearing misfolded proteins. Accordingly, pharmacological or genetic inhibition of autophagy prevented Zn(II)-mediated mutp53H175 degradation as well as the ability of the Zn(II) compound to restore wtp53 DNA-binding and transcription activity from this mutant. By contrast, inhibition of the proteasome failed to do so, suggesting that autophagy is the main route for p53H175 degradation. Mechanistically, Zn(II) restored the wtp53 ability to induce the expression of the p53 target gene DRAM (damage-regulated autophagy modulator), a key regulator of autophagy, leading to autophagic induction. Accordingly, inhibition of wtp53 transactivation by pifithrin-α (PFT-α) impaired both autophagy and mutp53H175 degradation induced by curcumin-based zinc compound (Zn(II)-curc). Viewed together, our results uncover a novel mechanism employed by Zn(II)-curc to reactivate mutp53H175, which involves, at least in part, induction of mutp53 degradation via wtp53-mediated autophagy.

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.

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. 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. 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. 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.

Autophagy is a catabolic process whose activation may help cancer cells to adapt to cellular stress although, in some instances, it can induce cell death. Autophagy stimulation or inhibition has been considered an opportunity to treat cancer, especially in combination with anticancer therapies, although autophagy manipulation may be viewed as controversial. Thus, whether to induce or to inhibit autophagy may be the best option in the different cancer patients is still matter of debate. Her we will recapitulate the possible advantages or disadvantages of manipulating autophagy in cancer, not only with the aim to obtain cancer cell death and disable oncogenes, but also to evaluate its interplay with the immune response which is fundamental for the success of anticancer therapies.

Understanding the mechanisms of autophagy induction and its role during chemotherapeutic treatments is of fundamental importance in order to manipulate it to improve the outcome of chemotherapy. In particular whether the bortezomib-induced autophagy plays a pro-survival or pro-death role is still controversial. In this study we investigated if bortezomib induced endoplasmic reticulum (ER) stress and activated autophagy in Primary Effusion Lymphoma (PEL) cells and how they influenced cell survival. We found that bortezomib induced up-regulation of the pro-survival and pro-death ER stress molecules BIP and CHOP and activated c-Jun NH2-terminal kinase (JNK), resulting in Bcl-2 phosphorylation and induction of autophagy. JNK and autophagy activation played a pro-survival role in this setting, thus their inhibition increased the bortezomib cytotoxic effect and PARP cleavage in PEL cells. Based on our results we suggest that the combination of bortezomib with JNK or autophagy inhibitors could be exploited to improve the outcome of therapy of this aggressive B cell lymphoma.

Optimal tumor eradication often results from the death of malignant cells, as induced by chemotherapeutic agents, coupled to the induction of antitumor immune responses. However, cancer cells frequ...