**5. Closing remarks**

### **5.1 Wnt/β-catenin pathway: radio-protective role and effect in RT-induced salivary gland damage**

In irradiation studies and radioprotection literature, numerous cellular signaling pathways and cell-cycle alteration mechanisms have been explored. Of those, the Wnt/β-catenin signaling pathway seems to receive the utmost attention, recently, towards preventing the damage caused by irradiation [119]. Briefly, this canonical Wingless–Int (Wnt) pathway leads to the accumulation and translocation of co-activator β-catenin, a multi-functional protein involved in cell–cell adhesion, gene transcription and physiologic homeostasis (adullt), into the nucleus, via a series of molecular events initiated through the binding of specific Wnt proteins to the frizzled receptors on the cell surface. The pathway plays a critical role in cell regulating cell migration and determining cell fate, and mutations have been linked to human birth defects, cancer and other disorders and diseases [120–123].

Activating the canonical Wnt/β-catenin signaling pathway is complex. It depends on a family of glyco-proteins involved in cell-to-cell communication. To simplify, the interaction of ß-catenin with the cell adhesion molecule, e-cadherin, is involved in phenotypes: adhesion, mobility and proliferation [121, 122]. In absence of a Wnt ligand, β-catenin is degraded by the "destruction complex". Several proteins are involved within this complex whereby Axin acts as a scaffold protein facilitating the interaction of Glycogen Synthase Kinase 3β (GSK-3β), Adenomatous Polyposis Coli (APC) and Casein Kinase 1α (CK1α), for β-catenin phosphorylation [123, 124]. Then, phosphorylated β-catenin is recognized by the β-transducin-repeat-containing protein (β-TrCP) and goes through the ubiquitin-proteasome degradation pathway. When the Wnt ligand activates Wnt signaling through the plasmatic membrane receptor frizzled with other lipoprotein receptors, the cytoplasmic protein disheveled (Dvl) is recruited and thereby activated. Herein, the activation of Dvl disrupts the "destruction complex" by dissociation of the GSK-3β from the Axin and inhibits the GSK-3β. As a result, β-catenin phosphorylation is also inhibited, allowing stabilization and translocation of β-catenin into the nucleus. Nuclear β-catenin then binds to a transcription factor-T cell factor and a lymphoid-enhancing factor (Tcf/Lef) and finally activates a response, *i.e.* changes in gene expression [120, 125, 126].

The Wnt signaling pathway cross-talks with other signaling pathways, and can be modulated by several activators and inhibitors. For example, the utilization of

growth factors, to activate or inhibit, has been extensively studied, further adding to the complexity given the wide range of involved genes [119]. Cross-talk between signaling pathways is possible via the common regulatory protein GSK-3β. For example, when the epidermal growth factor (EGF) is recognized by its native receptor (EGF-R), this complex activates the afore-mentioned phosphoinositide 3-kinase (PI3K) which facilitates the activation of AKT kinase regulator. Herein, the activation of AKT results in the inhibition of GSK-3β by phosphorylation [127–129] and ultimately leads to the translocation of β-catenin into the nucleus. On the other hand, the fibroblast growth factor (FGF) is also able to cross-talk with GSK-3β (common pathway with EGF) and the activation of its native receptor (FGF-R) is followed by PI3K which then results in the inhibition of GSK-3β via AKT activation [125, 130]. Herein, FGF-R activation also involves MapK activation which inhibits GSK-3β through the p90 ribosomal protein s6 kinase (p90rsk) in an AKT-independent manner [131–133]. Therefore, activating the Wnt signaling pathway (**Figure 4**) through the utilization of cytoplasmic regulatory proteins (from other signaling pathways) is potentially able to promote β-catenin stabilization, its translocation to the nucleus and the activation of survival genes [134]. Such understanding and revelations can lead to produce a plausible and innovative alternative strategy for the activation of native repair systems that may allow and promote the survival of the cells during and after RT. Possibly, can be even extended to explore plausibility for prevention.

To the best of knowledge, Hakim *et al.* [135] conducted one of the first/earliest *clinical* studies connecting signaling pathways (Wnt/β-catenin and TGF-β) with salivary gland irradiation damage. They reported an alteration in the expression pattern of Wnt1 in viable irradiated acinar cells of xerostomic patients, suggesting a possible therapeutic effect of the Wnt pathway in controlling RT-induced salivary gland damage and dysfunction [135], in accordance with previous *in vitro* studies [120]. Following this line of research, Hai *et al.* [136] carried out a study analyzing the transient activation of the Wnt/β-catenin signaling pathway to prevent

**Figure 4.** *EGF and FGF pathway(s) interaction with ß-catenin and canonical Wnt signaling pathway.*

#### *Salivary Gland Radio-Protection, Regeneration and Repair: Innovative Strategies DOI: http://dx.doi.org/10.5772/intechopen.94898*

irradiation damage to the salivary glands. They reported, using a murine model, that activating the Wnt/β-catenin pathway through the transient activation of Wnt1 in the basal epithelium helped to prevent chronic salivary dysfunction generated by local irradiation, specifically via suppressing apoptosis and preserving or rescuing the life of salivary stem/progenitor cells. Salivation in experimental mice when compared to controls (animals receiving only RT) was increased/higher [120, 136]. However, the radioprotective effect of Wnt/β-catenin activation seems, thus far, to only occur within a limited time lapse. Activating the signaling path 3 days before or 3 days after irradiation yielded dissimilar effects on the tissues [136].

Indeed, in another approach, the activation and modulation of cell signaling pathway(s) using a cocktail (more than one) of activators has been suggested, with the Wnt signaling pathway (and its components) as therapeutic target(s). Thula *et al.* [51] evaluated the effect of EGF and bFGF (basic FGF) in salivary gland explants, reporting promising results regarding gland radioprotection [51]. Overall, taking the studied findings into account, it can be proposed that a Wnt/β-catenin signaling pathway activator might be a good candidate to be developed as a potential preventive and therapeutic strategy against the RT-induced salivary gland damage. Herein, as was and is the present scenario with cells, proteins, genes, growth factors and drugs, a suitable delivery vehicle is once more, deemed vital.

**Technology Promise in Translational Tissue Engineering and NanoMedicine**the interplay between tissue engineering, regenerative medicine, biomaterials, bionanotechnology and nanomedicine continues to be the hallmark of current scientific research World-wide, promising to change every aspect of human life via creating revolutionary materials of biological origin for use in the diagnosis and treatment of devastating human diseases, a multi-disciplinary approach to innovative and translational solutions, suitable for scale-up, safe, efficacious and cost-effective routine clinical use [137–139]. Whether conventional small-molecule agents or emerging protein and/or peptide-based macromolecular biopharmaceutics, therapeutic effect is of vital significance. Controlled or at least predictable delivery is also substantially necessary. An intense effort is invested into engineering such complex bio-systems capable to achieve optimum cell-material interactions, while keeping intact the materials bulk properties. One of the core interests of nanobiotechnology, for example, this decade has been drug/gene/cell bio-functional delivery, driving the design and development of bio-inspired, intelligent or "smart" nano-systems [137, 138, 140]. It can be stated that a competitive and superiorly successful delivery system should offer: therapeutic outcome enhancement, patient compliance improvement and overall cost reduction of therapy. For HNC cases suffering RT-induced salivary gland damage and dysfunction, an attractive delivery system, for clinical ease-of-use, can perhaps entail a directly injectable formulation, sterilizable, capable to efficiently-hold a dose-responsive bio-load, maintain its bio-activity over time, and "predictably" control its pharmaco-kinetic release profile.

#### **Funding and acknowledgments**

This work was supported by generous funding and operating grants provided to the BioMAT'X R&D&I Group, part of CIIB (Centro de Investigación e Innovación Biomédica at UAndes), through the Faculty of Dentistry and Fondo de Ayuda a la Investigacion FAI - No. **INV-IN-2015-101** (2015–2019), Department for Research, Development and Innovation, Universidad de los Andes, Santiago de Chile. The authors wish to acknowledge supplementary funding provided under the awarded national grants from CORFO-CTecnológicos para la Innovación #**18COTE-89695** (the bioFLOSS project, 2018–2021) and CONICYT-FONDEF Chile **#ID16I10366** (the maxSALIVA project, 2016/17–2020).
