Alessia Garufi

Apoptosis as anticancer mechanism: function and dysfunction of its modulators and targeted therapeutic strategies

Apoptosis is a form of programmed cell death that results in the orderly and efficient removal of damaged cells, such as those resulting from DNA damage or during development. Apoptosis can be triggered by signals from within the cell, such as genotoxic stress, or by extrinsic signals, such as the binding of ligands to cell surface death receptors. Deregulation in apoptotic cell death machinery is an hallmark of cancer. Apoptosis alteration is responsible not only for tumor development and progression but also for tumor resistance to therapies. Most anticancer drugs currently used in clinical oncology exploit the intact apoptotic signaling pathways to trigger cancer cell death. Thus, defects in the death pathways may result in drug resistance so limiting the efficacy of therapies. Therefore, a better understanding of the apoptotic cell death signaling pathways may improve the efficacy of cancer therapy and bypass resistance. This review will highlight the role of the fundamental regulators of apoptosis and how their deregulation, including activation of anti-apoptotic factors (i.e., Bcl-2, Bcl-xL, etc) or inactivation of pro-apoptotic factors (i.e., p53 pathway) ends up in cancer cell resistance to therapies. In addition, therapeutic strategies aimed at modulating apoptotic activity are briefly discussed.

Degradation of mutant p53H175 protein by Zn(II) through autophagy

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.

JNK and Macroautophagy Activation by Bortezomib Has a Pro-Survival Effect in Primary Effusion Lymphoma Cells

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.

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

BackgroundMutations 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.MethodsP53 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.ResultsThe 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.ConclusionsOur 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.

Zinc supplementation is required for the cytotoxic and immunogenic effects of chemotherapy in chemoresistant p53-functionally deficient cells

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 frequently become resistant to the cytotoxic activity of chemotherapy. The aim of the present study was to evaluate whether zinc dichloride (ZnCl2), which was known to re-establish the chemosensitivity of cancer cells by reactivating p53, promotes immunogenic instances of cell death. We found that ZnCl2, in combination with chemotherapeutic agents such as cisplatin and adriamycin (ADR), favors the apoptotic demise of chemoresistant cells, while cisplatin and ADR alone fail to do so. The co-culture of immature dendritic cells (DCs) with cancer cells succumbing to the co-administration of chemotherapy and ZnCl2 led to DC activation, as indicated by the upregulation of the activation markers CD83 and CD86. In part, such process depended on cell death, as it was limited (but not abrogated) by the pan-caspase inhibitor Z-VAD-fmk. Moreover, DC activation relied on the ZnCl2-induced exposure of calreticulin (CRT) on the surface of cancer cells, correlating with the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), a marker of endoplasmic reticulum stress. The siRNA-mediated knockdown of CRT as well as the inhibition of CRT exposure with brefeldin A strongly impaired DC maturation, indicating CRT translocation as induced by that ZnCl2 is a key event in this setting. Altogether, these results suggest that ZnCl2, has the potential to enhance the therapeutic effects of antineoplastic agents not only by improving their cytotoxic activity but also by promoting CRT exposure.

Targeting COX-2/PGE2 Pathway in HIPK2 Knockdown Cancer Cells: Impact on Dendritic Cell Maturation

Background Homeodomain-interacting protein kinase 2 (HIPK2) is a multifunctional protein that exploits its kinase activity to modulate key molecular pathways in cancer to restrain tumor growth and induce response to therapies. For instance, HIPK2 knockdown induces upregulation of oncogenic hypoxia-inducible factor-1 (HIF-1) activity leading to a constitutive hypoxic and angiogenic phenotype with increased tumor growth in vivo. HIPK2 inhibition, therefore, releases pathways leading to production of pro-inflammatory molecules such as vascular endothelial growth factor (VEGF) or prostaglandin E2 (PGE2). Tumor-produced inflammatory mediators other than promote tumour growth and vascular development may permit evasion of anti-tumour immune responses. Thus, dendritic cells (DCs) dysfunction induced by tumor-produced molecules, may allow tumor cells to escape immunosurveillance. Here we evaluated the molecular mechanism of PGE2 production after HIPK2 depletion and how to modulate it. Methodology/Principal findings We show that HIPK2 knockdown in colon cancer cells resulted in cyclooxygenase-2 (COX-2) upregulation and COX-2-derived PGE2 generation. At molecular level, COX-2 upregulation depended on HIF-1 activity. We previously reported that zinc treatment inhibits HIF-1 activity. Here, zinc supplementation to HIPK2 depleted cells inhibited HIF-1-induced COX-2 expression and PGE2/VEGF production. At translational level, while conditioned media of both siRNA control and HIPK2 depleted cells inhibited DCs maturation, conditioned media of only zinc-treated HIPK2 depleted cells efficiently restored DCs maturation, seen as the expression of co-stimulatory molecules CD80 and CD86, cytokine IL-10 release, and STAT3 phosphorylation. Conclusion/Significance These findings show that: 1) HIPK2 knockdown induced COX-2 upregulation, mostly depending on HIF-1 activity; 2) zinc treatment downregulated HIF-1-induced COX-2 and inhibited PGE2/VEGF production; and 3) zinc treatment of HIPK2 depleted cells restored DCs maturation.

Glucose restriction induces cell death in parental but not in homeodomain-interacting protein kinase 2-depleted RKO colon cancer cells: molecular mechanisms and implications for tumor therapy

Tumor cell tolerance to nutrient deprivation can be an important factor for tumor progression, and may depend on deregulation of both oncogenes and oncosuppressor proteins. Homeodomain-interacting protein kinase 2 (HIPK2) is an oncosuppressor that, following its activation by several cellular stress, induces cancer cell death via p53-dependent or -independent pathways. Here, we used genetically matched human RKO colon cancer cells harboring wt-HIPK2 (HIPK2+/+) or stable HIPK2 siRNA interference (siHIPK2) to investigate in vitro whether HIPK2 influenced cell death in glucose restriction. We found that glucose starvation induced cell death, mainly due to c-Jun NH2-terminal kinase activation, in HIPK2+/+cells compared with siHIPK2 cells that did not die. 1H-nuclear magnetic resonance quantitative metabolic analyses showed a marked glycolytic activation in siHIPK2 cells. However, treatment with glycolysis inhibitor 2-deoxy-D-glucose induced cell death only in HIPK2+/+ cells but not in siHIPK2 cells. Similarly, siGlut-1 interference did not re-establish siHIPK2 cell death under glucose restriction, whereas marked cell death was reached only after zinc supplementation, a condition known to reactivate misfolded p53 and inhibit the pseudohypoxic phenotype in this setting. Further siHIPK2 cell death was reached with zinc in combination with autophagy inhibitor. We propose that the metabolic changes acquired by cells after HIPK2 silencing may contribute to induce resistance to cell death in glucose restriction condition, and therefore be directly relevant for tumor progression. Moreover, elimination of such a tolerance might serve as a new strategy for cancer therapy.

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

BackgroundMutations 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.Methodsp53 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.ResultsHere, 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.ConclusionThese 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.

High glucose dephosphorylates serine 46 and inhibits p53 apoptotic activity.

BackgroundIn response to diverse genotoxic stimuli p53 is activated as transcription factor to exert its tumor-suppressor function. P53 activation requires protein stabilization, nuclear localization and posttranslational modifications in key residues that may influence p53 selection of target genes. Among them, serine 46 (Ser46) phosphorylation is considered specific for apoptotic activation. Hyperglicaemia, the high blood glucose condition, may negatively affect tumor response to therapies through several mechanisms, conferring resistance to drug-induced cell death. However, whether high glucose might modify p53Ser46 phosphorylation has never been addressed.Methods and resultsHere, we performed biochemical and molecular analyses in different cancer cell lines treated with chemotherapy in the presence or absence of high glucose condition. Analyses of p53 posttranslational modifications showed that drug-induced p53Ser46 phosphorylation was reduced by high glucose. Such reduction depended by high glucose-induced calyculin A-sensitive phosphatase(s), able to specifically target p53Ser46 phosphorylation. The specific effect on Ser46 phosphorylation was addressed by analysing Ser15 phosphorylation that instead was not modified by high glucose. In agreement, a constitutively phosphorylated Ser46D p53 mutant was resistant to high glucose. As a consequence of phosphoSer46 impairment, high glucose reduced the tumor cell response to drugs, correlating with reduced p53 apoptotic transactivation. The drug-induced apoptotic cell death, reduced by high glucose, was finally restored by the phosphatase inhibitor calyculin A.ConclusionsThese data indicate that high glucose specifically inhibited Ser46 phosphorylation thus reducing p53 apoptotic activity. These results uncover a new mechanism of p53 inactivation providing an interesting novel molecular link between metabolic diseases such as diabetes or obesity and tumor progression and resistance to therapies.

Gentian violet induces wtp53 transactivation in cancer cells

Recent studies suggest that gentian violet (GV) may have anticancer activity by inhibiting for instance NADPH oxidases (Nox genes) whose overexpression is linked to tumor progression. Nox1 overexpression has been shown to inhibit transcriptional activity of the oncosuppressor p53, impairing tumor cell response to anticancer drugs. The tumor suppressor p53 is a transcription factor that, upon cellular stress, is activated to induce target genes involved in tumor cell growth inhibition and apoptosis. Thus, its activation is important for efficient tumor eradication. In this study, we examined the effect of GV on wild-type (wt) p53 activity in cancer cells. We found that GV was able to overcome the inhibitory effect of the NADPH oxidase Nox1 on p53 transcriptional activity. For the first time we show that GV was able to directly induce p53/DNA binding and transcriptional activity. In vitro, GV markedly induced cancer cell death and apoptotic marker PARP cleavage in wtp53-carrying cells. GV-induced cell death was partly inhibited in cells deprived of p53, suggesting that the anticancer activity of GV may partly depend on p53 activation. GV is US Food and Drug Administration approved for human use and may, therefore, have therapeutic potential in the management of cancer through p53 activation.