Quercetin as an Emerging Anti-Melanoma Agent: A Four-Focus Area Therapeutic Development Strategy

Replacing current refractory treatments for melanoma with new prevention and therapeutic approaches is crucial in order to successfully treat this aggressive cancer form. Melanoma develops from neural crest cells, which express tyrosinase -- a key enzyme in the pigmentation pathway. The tyrosinase enzyme is highly active in melanoma cells and metabolizes polyphenolic compounds; tyrosinase expression thus makes a feasible a target for polyphenol-based therapies. For example, quercetin (3,3′,4′,5,7-pentahydroxyflavone) is a highly ubiquitous and well-classified dietary polyphenol found in various fruits, vegetables and other plant products including onions, broccoli, kale, oranges, blueberries, apples, and tea. Quercetin has demonstrated anti-proliferative and pro-apoptotic activity in various cancer cell types. Quercetin is readily metabolized by tyrosinase into various compounds that promote anti-cancer activity; additionally, given that tyrosinase expression increases during tumorigenesis, and its activity is associated with pigmentation changes in both early- and late-stage melanocytic lesions, it suggests that quercetin can be used to target melanoma. In this review we explore the potential of Quercetin as an anti-melanoma agent utilizing and extrapolating on evidence from previous in vitro studies in various human malignant cell lines and propose a four-focus area strategy to develop quercetin as a targeted anti-melanoma compound for use as either a preventative or therapeutic agent. The four areas of focus include utilizing quercetin to i) modulate cellular bioreduction potential and associated signaling cascades, ii) affect transcription of relevant genes, iii) regulate epigenetic processes, and iv) develop effective combination therapies and delivery modalities/protocols. In general, quercetin could be used to exploit tyrosinase activity to prevent, and/or treat, melanoma with minimal additional side effects

Pharmacological Activation of the EDA/EDAR Signaling Pathway Restores Salivary Gland Function following Radiation-Induced Damage

Radiotherapy of head and neck cancers often results in collateral damage to adjacent salivary glands associated with clinically significant hyposalivation and xerostomia. Due to the reduced capacity of salivary glands to regenerate, hyposalivation is treated by substitution with artificial saliva, rather than through functional restoration of the glands. During embryogenesis, the ectodysplasin/ectodysplasin receptor (EDA/EDAR) signaling pathway is a critical element in the development and growth of salivary glands. We have assessed the effects of pharmacological activation of this pathway in a mouse model of radiation-induced salivary gland dysfunction. We report that post-irradiation administration of an EDAR-agonist monoclonal antibody (mAbEDAR1) normalizes function of radiation damaged adult salivary glands as determined by stimulated salivary flow rates. In addition, salivary gland structure and homeostasis is restored to pre-irradiation levels. These results suggest that transient activation of pathways involved in salivary gland development could facilitate regeneration and restoration of function following damage

Administration of growth factors promotes salisphere formation from irradiated parotid salivary glands

Worldwide, 500,000 cases of head and neck cancer (HNC) are reported each year and the primary treatment for HNC is radiotherapy. Although the goal of radiotherapy is to target the tumor, secondary exposure occurs in surrounding normal tissues, such as the salivary glands. As a result, despite successful treatment of the cancer, patients are left with long-term side effects due to direct damage to the salivary glands. The effect is chronic and currently there is no treatment. Stem cells are an attractive therapeutic option for treatment of radiation-induced glandular dysfunction because of the potential to regenerate damaged cell populations and restore salivary gland function. However, limited knowledge about the endogenous stem cell population post irradiation hinders the development for stem cell based therapies. In this study, an ex vivo sphere formation cell culture system was utilized to assess the self-renewal capacity of cells derived from parotid salivary glands at a chronic time point following radiation. Salivary glands from irradiated mice generate significantly fewer salispheres, but can be stimulated with fetal bovine serum (FBS) to generate an equivalent number of salispheres as unirradiated salivary glands. Interestingly, the number and size of salispheres formed is dependent on the concentration of FBS supplemented into the media. Salispheres derived from irradiated glands and cultured in FBS media were found to contain cells that proliferate and express progenitor and acinar cell markers such as Keratin 5, Keratin 14, Aquaporin 5, and NKCC1. Utilization of insulin-like growth factor (IGF1) injections following radiation treatment restores salivary gland function and improves salisphere generation. These findings indicate that stimulation of these cellular populations may provide a promising avenue for the development of cell-based therapies for radiation induced salivary gland damage.[R01 DE023534]Open access journal.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Label-Retaining Cells in the Adult Murine Salivary Glands Possess Characteristics of Adult Progenitor Cells

<div><p>Radiotherapy is the primary treatment for patients with head and neck cancer, which account for roughly 500,000 annual cases worldwide. Dysfunction of the salivary glands and associated conditions like xerostomia and dysphagia are often developed by these patients, greatly diminishing their life quality. Current preventative and palliative care fail to deliver an improvement in the quality of life, thus accentuating the need for regenerative therapies. In this study, a model of label retaining cells (LRCs) in murine salivary glands was developed, in which LRCs demonstrated proliferative potential and possessed markers of putative salivary progenitors. Mice were labeled with 5-Ethynyl-2′-deoxyuridine (EdU) at postnatal day 10 and chased for 8 weeks. Tissue sections from salivary glands obtained at the end of chase demonstrated co-localization between LRCs and the salivary progenitor markers keratin 5 and keratin 14, as well as <i>kit</i> mRNA, indicating that LRCs encompass a heterogeneous population of salivary progenitors. Proliferative potential of LRCs was demonstrated by a sphere assay, in which LRCs were found in primary and secondary spheres and they co-localized with the proliferation marker Ki67 throughout sphere formation. Surprisingly, LRCs were shown to be radio-resistant and evade apoptosis following radiation treatment. The clinical significance of these findings lie in the potential of this model to study the mechanisms that prevent salivary progenitors from maintaining homeostasis upon exposure to radiation, which will in turn facilitate the development of regenerative therapies for salivary gland dysfunction.</p></div

Differentiation of Salivary gland Spheres.

<p>A–B) Amylase staining (red) of parotid-derived spheres at days 2–3 in culture. C–D) Confocal images at Z = 0.5 um and Z = 6 um of double staining for amylase (red) and EdU (green) at day 4. Areas in yellow dashed squares are shown in C’ and D’. White arrow points at an amylase-positive cell with traces of EdU. Glands were obtained from mice at 10 weeks of age. E–G) Double immunofluorescence staining for Amylase (red) and EdU (Green) of parotid gland of 10-week old mice. White arrowhead points at LRCs in the acinar compartment; yellow arrowhead points at LRCs in ductal structures.</p

Sphere Assay with murine salivary glands.

<p>Representative microscope images of spheres grown from submandibular (A) and Parotid (B) glands from 10-week old mice.</p

Molecular markers in salivary gland LRCs.

<p>Representative images of parotid and submandibular glands of 10-day old and 10-week old animals stained for Keratin 14 (A–D), Keratin 5 (E–L), Smooth Muscle alpha Actin (M–P). Q–T) Fluorescence in Situ Hybridization for kit mRNA. EdU LRCs are shown in green, DAPI in blue, and all other markers in red. White arrowheads point at co-localization of each marker with the LRCs in the acinar compartment. Yellow arrowheads point at co-localization of each marker with the LRCs in the ductal compartment. Full size images of every panel are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107893#pone.0107893.s002" target="_blank">Figure S2</a>.</p

Label retaining assay in murine salivary glands.

<p>A) Label Retaining Assay. At 10 days after birth (P10), FVB mice were pulsed with 4 doses of EdU (100 mg/kg) or BrdU (30 mg/kg) 12 hours apart. Tissue was collected from 10-week old animals. B) LRCs from 3 mice were manually quantified per salivary gland compartment (acinar and ductal). Data are expressed as percentage from the total of cells in each individual compartment for both parotid and submandibular glands. C) Comparison of labeling efficiency between EdU and BrdU. Data are shown as percentage of LRCs per individual compartment. A 2-sided unpaired T-test was performed for analysis (n = 5 per group). D–E) Representative images of parotid and submandibular glands of BrdU-pulsed animals. LRCs in the acinar compartment (a) are shown with white arrowheads. LRCs in ductal compartment (d) are pointed with black arrows. Example of ductal compartment is delineated by dashed line and pointed with black arrowhead F–K) Representative fluorescent images of salivary glands from EdU-pulsed animals. EdU LRCs are shown in green and DAPI in blue.</p

Effect of radiation on salivary gland LRCs.

<p>A) Experimental setup. A single 5 Gy dose of radiation was given at week 4 to EdU-pulsed animals (n = 7). Tissue was collected at week 10. Representative images of EdU staining of parotid (B) and submandibular (C) glands are shown for irradiated animals and untreated controls. EdU positive cells were quantified manually per individual compartment for both treatments and expressed as percentage of LRCs per compartment for both glands (D–E). P values were obtained with 2-sided unpaired t-test per compartment (n = 7 for irradiated groups, n = 12 for parotid untreated control group and n = 6 for submandibular untreated control group). F) Experimental setup. 5 Gy dose of radiation treatment was given 24-hours prior tissue collection to EdU-pulsed animals (n = 3). G–J) Immunofluorescence staining of Activated Caspase-3 (red) and EdU (green) in parotid and submandibular glands. No co-localization is observed.</p

Administration of growth factors promotes salisphere formation from irradiated parotid salivary glands

<div><p>Worldwide, 500,000 cases of head and neck cancer (HNC) are reported each year and the primary treatment for HNC is radiotherapy. Although the goal of radiotherapy is to target the tumor, secondary exposure occurs in surrounding normal tissues, such as the salivary glands. As a result, despite successful treatment of the cancer, patients are left with long-term side effects due to direct damage to the salivary glands. The effect is chronic and currently there is no treatment. Stem cells are an attractive therapeutic option for treatment of radiation-induced glandular dysfunction because of the potential to regenerate damaged cell populations and restore salivary gland function. However, limited knowledge about the endogenous stem cell population post irradiation hinders the development for stem cell-based therapies. In this study, an ex vivo sphere formation cell culture system was utilized to assess the self-renewal capacity of cells derived from parotid salivary glands at a chronic time point following radiation. Salivary glands from irradiated mice generate significantly fewer salispheres, but can be stimulated with fetal bovine serum (FBS) to generate an equivalent number of salispheres as unirradiated salivary glands. Interestingly, the number and size of salispheres formed is dependent on the concentration of FBS supplemented into the media. Salispheres derived from irradiated glands and cultured in FBS media were found to contain cells that proliferate and express progenitor and acinar cell markers such as Keratin 5, Keratin 14, Aquaporin 5, and NKCC1. Utilization of insulin-like growth factor (IGF1) injections following radiation treatment restores salivary gland function and improves salisphere generation. These findings indicate that stimulation of these cellular populations may provide a promising avenue for the development of cell-based therapies for radiation-induced salivary gland damage.</p></div