Abstract: The dynamic and multiple functions of p53, together with its involvement in the most common non-infectious diseases, underscore the need to elucidate the complexity of the p53 regulatory networks. Pathological conditions such as cancer, neurodegeneration, ischemia, cholestasis, and atherosclerosis are all strongly associated with deregulated levels of apoptosis in which p53 dysfunction has a prominent role. We will highlight recent developments of p53-induced apoptosis in human diseases, with a focus on modulation of liver cell apoptosis. In addition, we will discuss controversies arising from widespread p53 activation as a therapeutic approach to cancer. Recent studies have provided relevant and unprecedented information about mechanistic antiapoptotic functions of the endogenous bile acid, ursodeoxycholic acid (UDCA), suggesting that the finely tuned, complex control of p53 by Mdm-2 (mouse double minute-2, an oncoprotein) is a key step in UDCA modulation of p53-triggered apoptosis. We will also review recent therapeutic strategies and clinical applications of targeted agents, their safety, and efficacy, with particular emphasis on potential benefits of UDCA.
Mammalian p53 has evolved from an ancient metazoan transcription factor, whose primordial function was to coordinate transcriptional responses to stress. The role of p53 as a tumor suppressor is likely to be a relatively recent adaptation of long-lived organisms to face the continuous accumulation of mutations resulting from substantial regenerative capacity. The importance of p53 in maintaining genome stability is exemplified by the finding that approximately half of all human tumors carry mutant p53. Currently, there are > 10 million people with tumors that contain inactivated p53, while a similar number have tumors in which the p53 pathway is partially abrogated by inactivation of other signaling components (Brown et al., 2009). However, it is clear that this protein also influences other aspects of health and disease apart from cancer development. Indeed, p53 exhibits diverse and global functions, in which cell cycle arrest, senescence, and apoptosis are the best characterized. Through these pathways, p53 facilitates the repair and survival of damaged cells or eliminates severely injured cells from the replicative pool to protect the organism. The dynamic and multiple functions of p53, together with its involvement in the modern world’s most common non-infectious diseases, highlight the importance in understanding its function within multiple fields, including toxicology and pharmacology.
Ursodeoxycholic acid (UDCA) is an endogenous hydrophilic bile acid in clinical use for the treatment of certain liver diseases. There is now strong evidence that the cytoprotective effects of this molecule result, in part, from its recognized antiapoptotic properties involving modulation of classical mitochondrial pathways. Interestingly, p53 has also been suggested to be involved in modulation of apoptosis by UDCA. In this regard, the use of UDCA as an agent to treat non-liver diseases associated with increased levels of apoptosis, such as neurodegenerative disorders, is now a major consideration.
Herein, we will provide a brief overview of the most recent developments involving p53 and apoptosis in disease pathogenesis, with a particular focus on liver. In addition, we will review the role of bile acids as modulators of general and p53-specific apoptosis. Finally, we will discuss possible controversies arising from targeting p53 in disease, and highlight the potential benefits of UDCA as a novel therapeutic agent for p53- and apoptosis-related disorders.
The Complexity of p53 Activation
p53 is undoubtedly one of the most studied proteins to date; yet, there is still much to know about its activities within the cell. p53 is at the center of a intricate protein network determining a multitude of important cellular responses that may vary from protecting the integrity of the genome, inducing apoptosis, regulating glycolysis and autophagy, to even promoting cell differentiation.
The p53 tumor suppressor is commonly described as a sequence-specific transcription factor that is kept at low levels in healthy cells. In addition to its complex tetrameric structure (reviewed in Joerger and Fersht, 2008), the activation profile of p53 is also multifaceted, at times unpredictable and unquestionably determined to control cell fate. Engagement of the p53 signaling pathway occurs in response to a broad range of stressors, intrinsic and extrinsic to the cell, which stabilize and affect p53 by a series of post-translational modifications. Phosphorylation is classically regarded as the first crucial step of p53 stabilization. There are a number of kinases responsible for p53 phosphorylation, such as the ataxia-telangiectasia mutated (ATM) kinase, Chk1/Chk2, JNK, p38, and others. Ubiquitination and acetylation of lysine residues are mutually exclusive modifications. Purified acetylated p53 cannot be ubiquitinated by its specific repressor mouse double minute-2 (Mdm-2) oncoprotein in vitro, and ubiquitination is significantly reduced upon induction of acetylation (Li et al., 2002). Finally, methylation, sumoylation, and neddylation have also been associated with regulation of p53 protein stability and transcriptional activation. The ability of p53 to integrate each kind of post-translational modification allows it to adequately respond by modulating the expression of different subsets of target genes (Riley et al., 2008).
Regulation of p53 function has traditionally been described at the level of transcription, translation, and structural alterations. However, there is general agreement that the key mechanism by which p53 is regulated is through control of protein stability, a mechanism primarily mediated by Mdm-2. Curiously, Mdm-2 is itself a transcriptional target of p53, therefore generating an autoregulatory negative feedback loop in which p53 activates the expression of its own inhibitor. To date, it has been demonstrated that the Mdm-2 protein can inhibit p53 by regulating its stability, cellular localization, and transactivation (Brooks et al., 2007). Mdm-2 functions as an E3 ubiquitin ligase targeting p53, and Mdm-2 itself, for proteasomal degradation. The ubiquitin ligase activity of Mdm-2 also contributes to the efficient nuclear export of p53. Finally, binding of Mdm-2 to the N-terminal transactivation domain of p53 may also preclude the interaction of p53 with transcriptional activators, namely p300, thus inhibiting the ability of p53 to activate transcription of target genes.
There are multiple layers of p53 regulation by Mdm-2. For instance, the tumor suppressor ADP-ribosylation factor (ARF) interferes with the Mdm-2/p53 interaction leading to p53 stabilization and activation (Stott et al., 1998). Another prominent regulator of Mdm-2 activity is Mdm-X; both proteins interact with each other to destabilize p53 (Uldrijan et al., 2007). Mutational inactivation of the retinoblastoma protein (pRb) is an alternate pathway of p53 activation. Unphosphorylated pRb is typically bound to the E2F-1 transcription factor, and functions as a growth inhibitory form of the protein, while hyperphosphorylated pRb mediates cell proliferation (Dyson, 1998). Inactivation of pRb releases transcription factor E2F-1, which can result in apoptosis by both p53-dependent and -independent mechanisms.
Curiously, p53 is not alone in its ability to regulate a myriad of cell functions. Almost two decades after the discovery of p53, two p53-homologs, p63 and p73 were described. The 60% homology with the p53 DNA binding domain allows p63 and p73 to regulate p53 target genes and, similar to p53, induce cell cycle arrest and apoptosis (Pietsch et al., 2008). Nevertheless, their primary functions are quite distinct.
Mechanisms of p53-dependent Apoptosis
One of the most dramatic responses to p53 activation is the induction of apoptosis. In hepatocytes, as well as in many other cell types, apoptosis occurs through either one of two major pathways described as either the intrinsic mitochondrial or extrinsic death receptor pathway (Kroemer et al., 2007). In the mitochondrial pathway, death stimuli target mitochondria either directly or through transduction by proapoptotic members of the Bcl-2 family, such as Bax and Bak. The mitochondria then release apoptogenic proteins, ultimately leading to caspase activation and apoptosis. In the death receptor pathway, and following interaction with its cognate ligand, the receptors located at the cellular membrane recruit adaptor proteins such as initiator caspase-8, triggering the activation of caspases to orchestrate apoptosis. The crosstalk between both pathways is mediated via Bid, and probably other factors that mediate cell death by modulation of both, and perhaps other unrecognized pathways.
Cells committed to die via p53-dependent apoptosis typically follow the mitochondrial pathway, although p53 can also modulate cell death through death receptors. Furthermore, most evidence suggests that the key contribution of p53 to apoptosis is primarily dependent on transcriptional activity. p53 has the ability to activate transcription of various proapoptotic genes, including those encoding members of the Bcl-2 family, such as the BH-3 only proteins Bax, Noxa, and Puma (Figure 1). The importance of Puma and Noxa to p53-mediated apoptosis became obvious when it was reported that certain cell types from puma knockout mice showed almost complete impairment to apoptosis via p53 (Jeffers et al., 2003). In other cells, apoptosis was only partially affected, and Noxa appeared to contribute to this effect. Alternatively, p53 can also trigger apoptosis by repression of antiapoptotic genes, such as survivin, thus promoting caspase activation (Hoffman et al., 2002).
Acting on the death receptor pathway of apoptosis, and in addition to stimulating fas transcription in the spleen, thymus, kidney, and lung (Bouvard et al., 2000), p53 overexpression enhances cell surface levels of Fas by promoting its trafficking from Golgi complex (Bennett et al., 1998). In addition, p53 activates DR5, the death domain-containing receptor for TRAIL; DR5 is induced in response to DNA damage (Wu et al., 1997) and, in turn, promotes cell death through caspase-8. Genes for proapoptotic proteins that may link apoptotic pathways, such as bid and the p53-induced protein with a death domain (PIDD), were also described as transcriptional targets of p53. Importantly, p53 is also involved in the activation of the apoptosome via induction of Apaf-1 expression (Moroni et al., 2001).
Despite the extent of its transcriptional activities, transcription-independent proapoptotic functions of p53 are emerging as an intensive area of research, which was largely initiated upon identifying the cytoplasm and/or mitochondria as novel sites of p53 action (reviewed in Speidel, 2010). Activation of p53 in enucleated cytoplasts is sufficient to directly or indirectly trigger apoptosis by inducing proapoptotic Bcl-2 family members (Schuler et al., 2000). A stress-stabilized cytoplasmic pool of p53 is probably the major source for p53 mitochondrial translocation; however, it is still a matter of debate on how the pool is generated. Some authors defend that unstressed cytoplasm contains a mixture of unstable polyubiquitinated p53 that is almost immediately targeted for proteasomal degradation, and functional monoubiquitinated p53 that serves as a source for p53 translocation to mitochondria (Figure 1). In the mitochondrion, p53 induces Bax and Bak oligomerization, physically interacts with protective Bcl-XL and Bcl-2, antagonizing their antiapoptotic effects, and also forms a complex with cyclophilin D leading to disruption of mitochondrial structure (Wolff et al., 2008) (Figure 1).
In addition to mitochondrial-targeted p53 actions, an alternative cytosolic p53 death pathway was recently reported that directly activates cytosolic Bax in UV-treated transformed mouse embryonic fibroblasts (Chipuk et al., 2005). After stress, nuclear p53 induces transcription of puma, which in turn, liberates p53 from an inactive pre-existing soluble p53-Bcl-XL complex, via binding to Bcl-XL. The cytosolic p53 then induces homo-oligomerization of Bax, followed by Bax mitochondrial translocation (Figure 1). Cytosolic p53 may also modulate other mechanisms apart from apoptosis. As an example, cytosolic p53 operates at the mitochondria to repress autophagy (Tasdemir et al., 2008).
Altogether, the combined diversity of p53-mediated apoptosis is quite remarkable. Now, it is critical to apply this knowledge and begin to elucidate the in vivo significance of transcription-independent apoptotic p53 activities for tumor suppression. It is also crucial to further dissect how the diverse and complex functions of nuclear and non-nuclear p53 are integrated to make definitive cell-fate decisions.
Targeting p53 in Disease
p53 is especially attractive for the development of new therapies in cancer, given the significant proportion of tumors with mutated p53 gene. In this respect, several approaches are being pursued for restoring p53 function in both mutant p53- and wild-type p53 tumors. In principal, functional activity of p53 mutants could be restored by introducing a copy of the wild-type p53 gene by a suitable viral vector; while wild-type, but inactive p53, could be activated by targeting Mdm-2 ubiquitination and interaction between p53 and its repressor.
The main strategies for either restoring p53 function or delivering exogenous therapeutic p53 have been extensively investigated, both in vitro and in vivo, particularly for treatment of hepatocellular carcinoma (HCC). At the present, viral vectors are the most promising p53 delivery agents for the treatment of HCC, and they have already been employed in several clinical trials (Tian et al., 2009). Nevertheless, gene therapy is currently limited to local administration, since efficient methods for systemic targeted delivery of viral vectors to tumor cells are still unavailable. Furthermore, significant enhancement in the gene transfer efficiency is desirable for improved HCC applications.
More recently, the design and development of small-molecular inhibitors of the Mdm-2/p53 interaction has emerged as a promising and novel cancer therapeutic strategy. Nutlin-3 and MI-219 are already in preclinical development or early phase clinical trials. Both display a high binding affinity and specificity to Mdm-2, a strong activity in cancer cells with wild-type p53, and a highly desirable pharmacokinetic profile (Shangary and Wang, 2009).
Nevertheless, the role of p53 remains controversial and its functions go well beyond malignant progression. The most effective means of treating cancer also carry severe and undesirable side effects, many times as a result of widespread p53 activation. Although some tumors no longer undergo p53-mediated cell death, apoptosis continues to contribute to chemotherapy- and/or radiation-induced damage of sensitive normal tissues (Gudkov and Komarova, 2003). Therefore, it has been proposed that temporary suppression of p53 during treatment of p53-deficient tumors could prevent this side effect. A recent study using a switchable-p53 mouse model revealed that delaying the activation of p53 until after the DNA-damage response by other DNA-repair mechanisms protected mice from negative effects of p53-induced apoptosis, normally observed in radiosensitive tissues (Christophorou et al., 2006).
Thus, drugs that turn off p53 function during, or shortly after, genotoxic injury may help to alleviate the side effects of cancer therapy while still retaining the tumor suppressor activity that is induced by unregulated oncogene action. This strategy has recently been tested experimentally through the use of a small molecule inhibitor of p53, pifithrin-α (PFT-α), which inhibits apoptosis in cells undergoing rapid p53-dependent cell death in response to various treatments, including γ radiation. Remarkably, the use of PFT-α failed to induce a marked carcinogenic response, possibly because p53 inhibition occurs only during the period of drug application (Komarov et al., 1999). Thus, in the absence of apoptosis, p53 might act as a survival factor that reduces the rate of mitotic catastrophe after radiation by keeping cells in prolonged growth arrest, thereby facilitating DNA repair. p53 inhibition could therefore improve the therapeutic outcome of radiation therapy not only by reducing damage to normal tissues, but also by potentiating tumor susceptibility to treatment.
In diseases other than cancer, the therapeutic inhibition of p53 may represent a new area for intervention. Induction of p53 during ischemia, for example, has been shown to contribute to tissue damage through activation of apoptosis. In contrast, temporary inhibition of p53 function might be highly beneficial in the prevention of injury to diverse organs (Georgiev et al., 2006), or in treatment of myocardial infarction (Matsusaka et al., 2006). In addition, treatment with S. miltiorrhiza, a traditional herbal medicine, was also shown to induce cytoplasmic sequestration of p53, down regulation of Bax, and up regulation of Bcl-2 protein in a rat model of cholestasis, supporting a role for p53 targeting in clinical management of hepatic disease induced by toxic bile salts (Oh et al., 2002). Finally, the tumor suppressor is also associated with neurodegenerative pathological conditions, including AIDS-associated neurodegeneration, stroke, and Parkinson’s, Alzheimer’s, and Huntington’s diseases (Vousden and Lane, 2007).
Role of Ursodeoxycholic Acid
UDCA is a hydrophilic bile acid widely used as a first choice treatment for patients with cholestatic liver diseases. Remarkably, the protective effects of UDCA are not confined to the liver, being also observed in nonhepatic cells (Rodrigues et al., 1998). Nevertheless, and despite its clinical efficacy, the precise mechanism by which UDCA ameliorates liver function is still not entirely clear. Most likely, UDCA functions as a pleiotropic molecule protecting cells through a coordinated process involving a variety of different pathways, including inhibition of apoptosis, protection of cholangiocytes against cytotoxic hydrophobic bile acids, and stimulation of impaired biliary secretion, depending on the type and stage of the disease (Castro et al., 2007). UDCA was also described as an important activator of survival pathways (Schoemaker et al., 2004), and regulator of the immune response (Miura et al., 2001).
It is well established that UDCA modulates the mitochondrial pathway of apoptosis (Rodrigues et al., 1999). In addition, UDCA reaches the nucleus where it might differentially regulate gene expression profiles. Indeed, microarray studies revealed that UDCA per se modulates the expression of at least 96 genes in primary rat hepatocytes, with most of them being involved in apoptosis and cell cycle regulation (Castro et al., 2005). Previous findings also demonstrated that UDCA modulates the TGF-β1-induced E2F-1/p53/Bax apoptotic pathway in primary rat hepatocytes, independent of its effect on mitochondria.
Consolidating these data, p53 was recently established as a key molecular target in UDCA prevention of cell death, showing that the finely tuned, complex control of p53 by Mdm-2 constitutes a prime target for UDCA modulation of p53-mediated hepatocyte death (Amaral et al., 2007). By inducing Mdm-2/p53 complex formation, UDCA reduces p53 activity, simultaneously blocking its transactivation domain and enhancing its export to cytosol. However, the precise mechanism by which UDCA induces Mdm-2/p53 binding is unclear.
The use of hydrophilic bile acids, such as UDCA, may represent a safe and efficient clinical therapy for diseases with higher susceptibility to apoptotic cell death, including p53-related disorders. Of note, although p53 is mainly considered a tumor suppressor protein, it is clear that loss of p53 does not always lead to increased tumor resistance to treatment. On the contrary, such a loss can actually contribute to chemo- and radiation sensitivity (Gudkov and Komarova, 2003). Nevertheless, it remains uncertain as to whether UDCA is capable of distinguishing “normal” from tumor cells, or even favors tumor development. Since the antiapoptotic function of UDCA is triggered by diverse toxic stimuli, such as hydrophobic bile acids, TGF-β1, or p53 overexpression, it is expectable that this bile acid acts in cells where p53 activation results in imbalanced apoptosis, but not in cancer cells where activated p53 counteracts excessive proliferation. More importantly, UDCA has been the first-line treatment of several liver diseases for decades, without increasing patients’ predisposition to cancer. Interestingly, recent studies have demonstrated a neuroprotective role of UDCA against neurotoxicity induced by the anticancer therapeutic drug, cisplatin, through downregulation of the p53 signaling pathway (Park et al., 2008). In fact, in addition to rat hepatocytes, p53 was already described as a target for bile acid’s cytoprotective functions in neuronal cells. Tauroursodeoxycholic acid (TUDCA) was shown to be a potent neuroprotective agent in experimental models of Huntington’s disease, ischemic and hemorrhagic stroke, and Parkinson’s disease (reviewed in Amaral et al., 2009). Preclinical data also suggest that other inhibitors of p53 may be effective therapeutic agents for neurodegenerative conditions (Culmsee and Mattson, 2005). Nevertheless, the clinical application of p53-modulating drugs is still under intense development and review.
It is well established that the p53 response is defective in most cancers, either by mutations or deletions in the p53 gene, or by alterations in the p53 pathway caused by other oncogenic events. These observations have raised a wide range of clinical possibilities both for diagnosis and treatment, rendering p53 an ideal target for anti-cancer drug design. Safety and efficacy of newly designed peptides or small molecules capable of modulating either wild-type or mutant p53 proteins are now being evaluated in patients enrolled in an expanding number of clinical trials. Hopefully, some of these molecules will become routinely applied, alone and in combination with established therapeutic interventions, to improving the clinical management of cancer patients.
Still, a key feature of p53 is that its role and function should always be considered to be significantly dependent on the cellular microenvironment. Ultimately, p53 may act to promote cell death or survival depending on the cell type, gene expression profile, protein activity, and the type of stress stimuli, among other criteria (Kim et al., 2009). This must always be considered when planning new strategies to target this tumor suppressor. In this respect, p53 has been implicated in other apoptosis-related disorders apart from cancer, such as atherosclerosis, ischemia, and neurodegenerative diseases, which are increasingly correlated with aging and stress of modern societies. It is noteworthy that just a slight constitutive hyperactivation of p53 induces an alarming premature aging phenotype in mice (Maier et al., 2004), thereby raising some concern for the use of p53 systemic activation in cancer therapy. The beneficial role of UDCA and/or TUDCA as fine-tuning modulators of deregulated apoptosis could perhaps represent a therapeutic option to overcome p53-mediated cell loss. This applies not only to cancer therapy, but also to other p53-associated disorders, thereby expanding the clinical applicability of these molecules.
Supported, in part, by grant PTDC/SAU-GMG/099162/2008 from Fundação para a Ciência e a Tecnologia (FCT), Lisbon, Portugal (to C.M.P.R). J.D.A. is a recipient of postdoctoral fellowship (SFRH/BPD/47376/2008) from FCT.
(Corresponding author: Cecília M. Rodrigues, Ph.D., iMed.UL, Research Institute for Medicines and Pharmaceutical Sciences, Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal.)
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