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Radiation-Induced Salivary Gland Dysfunction Results From p53-Dependent Apoptosis
Abstract
Radiotherapy for head-and-neck cancer causes adverse secondary side effects in the salivary glands and results in diminished quality of life for the patient. A previous in vivo study in parotid salivary glands demonstrated that targeted head-and-neck irradiation resulted in marked increases in phosphorylated p53 (serine(18)) and apoptosis, which was suppressed in transgenic mice expressing a constitutively active mutant of Akt1 (myr-Akt1).
Transgenic and knockout mouse models were exposed to irradiation, and p53-mediated transcription, apoptosis, and salivary gland dysfunction were analyzed.
The proapoptotic p53 target genes PUMA and Bax were induced in parotid salivary glands of mice at early time points after therapeutic radiation. This dose-dependent induction requires expression of p53 because no radiation-induced expression of PUMA and Bax was observed in p53-/- mice. Radiation also induced apoptosis in the parotid gland in a dose-dependent manner, which was p53 dependent. Furthermore, expression of p53 was required for the acute and chronic loss of salivary function after irradiation. In contrast, apoptosis was not induced in p53-/- mice, and their salivary function was preserved after radiation exposure.
Apoptosis in the salivary glands after therapeutic head-and-neck irradiation is mediated by p53 and corresponds to salivary gland dysfunction in vivo.
... Although radiotherapy is considered to be one of the main anticancer therapies for head and neck cancers, radiation-induced hyposalivation is also one of most common complications, mainly resulting from the destruction of salivary tissues with their high biological susceptibility to radiation [27,28]. Copious studies using a radiation model have been performed, with a variety of radiation dosages administered over different species [22,[28][29][30][31][32]. The irradiation procedure could be divided into single-dose and fractionated irradiation according to its schedules. ...
... Previous studies have reported that histological alterations such as acinar loss are intimately related to radiation dose [29,[34][35][36]. However, it should be considered that irradiation with a single high dose might significantly shorten the life span of the experimental animals [36]. ...
... These researchers reported that p53 could be a key molecule in the regulation of radiosensitivity via the probable involvement of DNA damage repair, cell cycle arrest, and apoptosis [28]. Noticeably, it was furthermore revealed that radiation exposure induced no apoptosis in knockout mice with p53 −/− as determined by immunohistochemistry of caspase-3 [29]. These findings demonstrated that post-irradiation apoptosis in the salivary gland could be mediated by p53. ...
Salivary secretory disorders are life-disrupting pathologic conditions with a high prevalence, especially in the geriatric population. Both patients and clinicians frequently feel helpless and get frustrated by the currently available therapeutic strategies, which consist mainly of palliative managements. Accordingly, to unravel the underlying mechanisms and to develop effective and curative strategies, several animal models have been developed and introduced. Experimental findings from these models have contributed to answer biological and biomedical questions. This review aims to provide various methodological considerations used for the examination of pathological fundamentals in salivary disorders using animal models and to summarize the obtained findings. The information provided in this review could provide plausible solutions for overcoming salivary disorders and also suggest purpose-specific experimental animal systems.
... Immunofluorescent staining for Yap using a total antibody that detects both phosphorylated and unphosphorylated proteins was performed to compare the percentage of cells that displayed nuclear Yap following radiation treatment. Cells within major ducts (excretory and striated ducts) have a distinctive response following radiation as evidenced by a lack of apoptosis at acute time points, a lack of induction of a compensatory proliferation response and their continued presence in clinical sections [11,[27][28][29][30]. Therefore, ductal cells were quantified separately and the remaining cellular populations were combined and labeled as the acinar compartment (Fig 1E-1G). ...
... Gy/day [5,29]. This study utilized a single dose of 5Gy, which is similar to the daily clinical fraction and sufficient to induce chronic salivary hypofunction [22,27,28,30], in order to investigate the kinetic events that underscore the damaged phenotype. In the mouse model utilized in this study, apoptosis peaks at 24-48 hours post-treatment leading to significant decreases in stimulated salivary flow rates as early as three days post-treatment [22,27,30]. ...
... This study utilized a single dose of 5Gy, which is similar to the daily clinical fraction and sufficient to induce chronic salivary hypofunction [22,27,28,30], in order to investigate the kinetic events that underscore the damaged phenotype. In the mouse model utilized in this study, apoptosis peaks at 24-48 hours post-treatment leading to significant decreases in stimulated salivary flow rates as early as three days post-treatment [22,27,30]. Decreased levels of apoptosis following radiation correlates with preserved stimulated salivary flow rates [27,30]. ...
Radiotherapy plays a major role in the curative treatment of head and neck cancer, either as a single modality therapy, or in combination with surgery or chemotherapy, or both. Despite advances to limit radiation-induced side-effects, the major salivary glands are often affected. This frequently leads to hyposalivation which causes an increased risk for xerostomia, dental caries, mucositis, and malnutrition culminating in a significant impact on patients’ quality of life. Previous research demonstrated that loss of salivary function is associated with a decrease in polarity regulators and an increase in nuclear Yap localization in a putative stem and progenitor cell (SPC) population. Yap activation has been shown to be essential for regeneration in intestinal injury models; however, the highest levels of nuclear Yap are observed in irradiated salivary SPCs that do not regenerate the gland. Thus, elucidating the inputs that regulate nuclear Yap localization and determining the role that Yap plays within the entire tissue following radiation damage and during regeneration is critical. In this study, we demonstrate that radiation treatment increases nuclear Yap localization in acinar cells and Yap-regulated genes in parotid salivary tissues. Conversely, administration of insulin-like growth factor 1 (IGF1), known to restore salivary function in mouse models, reduces nuclear Yap localization and Yap transcriptional targets to levels similar to untreated tissues. Activation of Rho-associated protein kinase (ROCK) using calpeptin results in increased Yap-regulated genes in primary acinar cells while inhibition of ROCK activity (Y-27632) leads to decreased Yap transcriptional targets. These results suggest that Yap activity is dependent on ROCK activity and provides new mechanistic insights into the regulation of radiation-induced hyposalivation.
... 4 Thus, the mechanism involved in the irradiation-induced loss of salivary gland function is a subject of great interest in the field, with clinical studies directed towards assessing therapies targeted to the recovery of cell function, prevention of functional loss, or regeneration of salivary glands. 5 The management strategies include stringent dental and oral hygiene and pharmacotherapies, such as salivary substitutes and sialagogues. Mandelonitrile beta-D-gentiobioside, which is commonly known as vitamin B17, is a natural cyanide-containing substance called nitriloside. ...
... 18 The chosen radiation dose was selected similar to Avila who used the same ionizing radiation dose and reported radiation-induced changes in the parotid gland of rats. 5 The results of the present study showed acinar degeneration, shrinkage and cytoplasmic vacuolization in group A (day 14 and day 30). Boraks, Tampelini, Pereira, and Chopard have linked acinar degeneration and vacuolization to the expansion of the endoplasmic reticulum which is associated with the cellular status that precedes apoptosis this expansion is related to compression of the nuclear material. ...
Background:
Radiotherapy is used as a treatment for head and neck cancers but increases the risk of salivary gland hypofunction. The management strategies include pharmacotherapies such as salivary substitutes and sialagogues which are largely temporary. In this study, we examine the regenerative potential of vitamin B17 to improve salivary gland function.
Objectives:
The present investigation aims to identify the effect of vitamin B17 (amygdaline) on the irradiated parotid salivary gland of albino rats.
Material and methods:
Twenty-eight adult male albino rats were randomly divided into two groups subjected to irradiation procedure. Fourteen were in the control group, receiving a daily 5 mL saline by oral gavage (7 rats for 14 days and 7 rats for 30 days) while the other fourteen were treated with a daily dose of vitamin B17 (grounded apricot kernel; GAK) at 400 mg/kg in 5 mL of saline by oral gavage (7 rats for 14 days and 7 rats for 30 days). The parotid glands were dissected from the two groups at 14 and 30 days from the day of exposure to irradiation. The parotid gland sections were subjected to H&E stain, immunohistochemical localization of epidermal growth factor (EGF) and PCR using transforming growth factor beta 2 (TGF-β2).
Results:
The histological abnormalities corroborate with the immunohistochemical localization of EGF and the PCR results of TGF β2, as their up-regulation in the control group demonstrate oxidative stresses and inflammation. The Treatment with GAK decreased oxidative stress and inflammation while promoting tissue regeneration.
Conclusions:
Vitamin B17 is a promising anti-inflammatory agent that boosts immunity, as the experimental group showed better histological architecture of the parotid gland than the other one.
... The submandibular glands (SMGs) produce >60% of total daily saliva secretion and account for most unstimulated saliva volume, whereas the parotid glands contribute to most stimulated saliva volume. 5,6 RT has been shown to result in significant and early loss of epithelial acinar cells, which are the predominant cells involved in saliva production, controlling both the volume and protein content in saliva. 2,5 The specific mechanisms resulting in acute and persistent xerostomia have not been elucidated and are hypothesized to be a result of macroscopic and microscopic changes to glandular tissue. ...
... 5,6 RT has been shown to result in significant and early loss of epithelial acinar cells, which are the predominant cells involved in saliva production, controlling both the volume and protein content in saliva. 2,5 The specific mechanisms resulting in acute and persistent xerostomia have not been elucidated and are hypothesized to be a result of macroscopic and microscopic changes to glandular tissue. ...
Objective
This study aimed to assess if Nigella sativa oil (NSO), a health supplement containing thymoquinone as a major component, can act as a protective agent in salivary gland stem cells following radiotherapy (RT) damage.
Methods
Forty, 10‐week‐old, male C3H/HeJ mice were randomized to four experimental groups: sham RT + H2O gavage (control) (N = 4); 15 Gy RT + H2O gavage (N = 12); sham RT + NSO gavage (N = 12); and 15 Gy RT + NSO gavage (N = 12). Weight changes, saliva production, and salivary gland histopathologic staining were recorded for each group over the course of the experiment.
Results
All mice in the sham RT + H2O gavage and sham RT + NSO gavage groups demonstrated 100% 60‐day survival. RT + H2O compared to RT + NSO gavaged mice were significantly underweight by an average of 6.4 g (p < .001). Salivary output showed significant decline in RT + H2O gavaged mice at days 3 and 16, whereas salivary output in RT + NSO during these same time periods was comparable to the control. At day 60, all mice that survived recovered salivary function regardless of their treatment arm. Salivary specimens from the RT + NSO gavage group demonstrated early signs of recovery of Kr 5+ salivary gland stem cells in both submandibular and sublingual glands at day 16 with complete recovery by day 60, marked by strong histopathologic staining, whereas the RT + H2O gavage group did not recover as effectively.
Conclusion
NSO may help preserve salivary function in mice treated with RT and may mitigate xerostomia by accelerating the recovery of salivary gland stem cells.
Level of evidence
Not applicable.
... Knowledge of the histopathological features of irradiated SGs derives from clinical observations, whereas most of our understanding of the cellular and molecular mechanisms involved in the loss of function and potential regenerative mechanisms are derived from animal studies. Loss of saliva from IR damage is due to the extensive depletion of acinar cells that fail to regenerate (392,393). Although the precise mechanisms that prevent acinar regeneration are unclear, three possible explanations are that IR 1) damages the acinar progenitor pool (394), 2) inhibits the process of acinar self-duplication that usually replaces acinar cells during homeostasis (395), or 3) creates a damaged microenvironment that prevents acinar regeneration. ...
... Expression of a constitutively active form of Akt (myr-Akt1) in the SGs inhibited apoptosis via reduced p53 phosphorylation and increased MDM2 phosphorylation in vivo (403). Consistently, mice lacking p53 showed significantly lower levels of apoptosis after IR compared with wild types, and salivary function was preserved (392). Presumably upstream of p53, FGF signaling via FGF10 binding to FGFR2 has been shown to activate the AKT/PI3K pathway, leading to increased activation of antiapoptotic molecules ultimately inhibiting apoptosis (404). ...
Salivary glands produce and secrete saliva, which is essential for maintaining oral health and overall health. Understanding both the unique structure and physiological function of salivary glands, as well as how they are affected by disease and injury will direct the development of therapy to repair and regenerate them. Significant recent advances, particularly in the OMICS field, increase our understanding of how salivary glands develop at the cellular, molecular and genetic levels; the signaling pathways involved, the dynamics of progenitor cell lineages in development, homeostasis and regeneration and the role of the extracellular matrix microenvironment. These provide a template for cell and gene therapies as well as bioengineering approaches to repair or regenerate salivary function.
... Furthermore, cell lysis and apoptosis as described in the granulation hypothesis is shown to be not significant early after radiation exposure [58,65,66]. More recently, it has been shown that expression of the p53 tumor-suppressor gene is required for both acute and chronic salivary gland dysfunction after radiation exposure, further suggesting that the granulation hypothesis alone does not explain salivary gland radiation sensitivity and that more complicated mechanisms are involved [67]. ...
... • Degranulation of serous cells and induction of nuclear aberrations the first day after irradiation • Recovery of tissue integrity observed from day 6 Zeilstra et al. [65] Male Wistar rats (9 weeks old) X-ray 15 Gy 1,0 Gy/min • Severe compromised gland function soon after irradiation, without significant cell loss • Later: loss of acinar cells accompanied by slow loss of gland function Paardekooper et al. [66] Male Wistar rats (10-11 weeks old) X-ray Up to 25 Gy 1,0 Gy/min • After 2 Gy dose: apoptotic cells observed until 24 h after treatment • 6 days after treatment: no apoptotic cells Avila et al. [67] Female Myr-Akt1 transgenic mice + p53 KO mice (4 weeks old) ...
At present, prostate cancer remains the second most occurring cancer in men, in Europe. Treatment efficacy for therapy of advanced metastatic disease, and metastatic castration-resistant prostate cancer in particular is limited. Prostate-specific membrane antigen (PSMA) is a promising therapeutic target in prostate cancer, seeing the high amount of overexpression on prostate cancer cells. Clinical investigation of PSMA-targeted radionuclide therapy has shown good clinical efficacy. However, adverse effects are observed of which salivary gland hypofunction and xerostomia are among the most prominent. Salivary gland toxicity is currently the dose-limiting side effect for PSMA-targeted radionuclide therapy, and more specifically for PSMA-targeted alpha therapy. To date, mechanisms underlying the salivary gland uptake of PSMA-targeting compounds and the subsequent damage to the salivary glands remain largely unknown. Furthermore, preventive strategies for salivary gland uptake or strategies for treatment of salivary gland toxicity are needed. This review focuses on the current knowledge on uptake mechanisms of PSMA-targeting compounds in the salivary glands and the research performed to investigate different strategies to prevent or treat salivary gland toxicity.
... Head and neck cancer patients routinely receive fractionated radiation treatment where the tumor region receives high radiation doses while salivary gland sparing techniques attempt to limit the radiation dose to is impairment of muscarinic receptor signaling (Coppes et al., 2000Konings et al., 2005) required for saliva formation and aquaporin channel activity required for fluid secretion (Takagi et al., 2003). Furthermore, Avila et al. (2009), have demonstrated that radiation also causes a significant reduction in saliva-secreting acinar cells due to p53-dependent apoptosis. Thus, the overall mechanism of radiation-induced salivary gland hypofunction likely involves perturbations in muscarinic receptor signaling, apoptosis of saliva-producing acinar cells and irreversible tissue damage. ...
... Consequently, chronic hyposalivation and changes in the saliva electrolyte composition occur along with a reduction in pH that leads to alterations in oral microbial flora, increased incidence of dental carries and oral infections and difficulties with swallowing, digestion, and speech(Hu et al., 2013;Pinna et al., 2015).Several groups have utilized rodent models to demonstrate that acute hyposalivation occurs immediately after IR, before the onset of overt gland damage, which is associated with sustained increases in the [Ca 2+ ] iLiu et al., 2013Liu et al., , 2017Ambudkar, 2018). In contrast, chronic IR-induced salivary dysfunction results from ROS production, increased caspase-3 activity, disruption of store-operated Ca 2+ entry (SOCE), cytoskeletal rearrangements, acinar cell apoptosis, sialadenitis and replacement of normal parenchyma with fibrotic tissue(Coppes et al., 2001;Radfar and Sirois, 2003;Teymoortash et al., 2005;Muhvic-Urek et al., 2006;Avila et al., 2009;Liu et al., 2013Liu et al., , 2017Wong et al., 2018). One of the early responses to IR Frontiers in Pharmacology | www.frontiersin.org ...
Although often overlooked in our daily lives, saliva performs a host of necessary physiological functions, including lubricating and protecting the oral cavity, facilitating taste sensation and digestion and maintaining tooth enamel. Therefore, salivary gland dysfunction and hyposalivation, often resulting from pathogenesis of the autoimmune disease Sjögren’s syndrome or from radiotherapy of the head and neck region during cancer treatment, severely reduce the quality of life of afflicted patients and can lead to dental caries, periodontitis, digestive disorders, loss of taste and difficulty speaking. Since their initial discovery in the 1970s, P2 purinergic receptors for extracellular nucleotides, including ATP-gated ion channel P2X and G protein-coupled P2Y receptors, have been shown to mediate physiological processes in numerous tissues, including the salivary glands where P2 receptors represent a link between canonical and non-canonical saliva secretion. Additionally, extracellular nucleotides released during periods of cellular stress and inflammation act as a tissue alarmin to coordinate immunological and tissue repair responses through P2 receptor activation. Accordingly, P2 receptors have gained widespread clinical interest with agonists and antagonists either currently undergoing clinical trials or already approved for human use. Here, we review the contributions of P2 receptors to salivary gland function and describe their role in salivary gland dysfunction. We further consider their potential as therapeutic targets to promote physiological saliva flow, prevent salivary gland inflammation and enhance tissue regeneration.
... Many research groups, including ours, have previously focused on examining the submandibular glands to assess the effectiveness of a tested experimental therapy against IR-induced injury (Yi et al. 2016;). However, examining parotids provided a better assessment of the therapy because 1) parotid glands have a major function during stimulated saliva secretion (de Almeida Pdel Grundmann et al. 2009), 2) parotids are more sensitive to IR than submandibular glands (Avila et al. 2009), and 3) salivary hypofunction and cell apoptosis in mouse parotids are correlated to IR doses (Humphries et al. 2006;Avila et al. 2009). Thus, this study examined the LSCE effect in parotid and submandibular glands. ...
... Many research groups, including ours, have previously focused on examining the submandibular glands to assess the effectiveness of a tested experimental therapy against IR-induced injury (Yi et al. 2016;). However, examining parotids provided a better assessment of the therapy because 1) parotid glands have a major function during stimulated saliva secretion (de Almeida Pdel Grundmann et al. 2009), 2) parotids are more sensitive to IR than submandibular glands (Avila et al. 2009), and 3) salivary hypofunction and cell apoptosis in mouse parotids are correlated to IR doses (Humphries et al. 2006;Avila et al. 2009). Thus, this study examined the LSCE effect in parotid and submandibular glands. ...
Stem cell–based therapies could provide a permanent treatment for salivary gland (SG) hypofunction caused by ionizing radiation (IR) injury. However, current challenges for SG stem cells to reach the clinic include surgical invasiveness, amount of tissue needed, cell delivery, and storage methods. The objective of this study was to develop a clinically less invasive method to isolate and expand human SG stem cells and then to obtain a cell-free extract to be used as a therapy for IR-injured SGs. Human labial glands were biopsied, and labial stem cells (LSCs) were expanded by explant culture. The LSC extract (LSCE) was obtained by releasing the cellular components after 3 freeze-thaw cycles and 17,000 g force centrifugation. LSCE was injected intravenously into mice that had their SGs injured with 13-Gy IR. Positive (non-IR) and negative (IR) control mice received injections of saline (vehicle control). Three pieces of labial glands (0.1 g weight) could expand 1 to 2 million cells. LSCs had a doubling time of 18.8 h; could differentiate into osteocytes, adipocytes, and chondrocytes; and were positive for mesenchymal stem cell markers. Both angiogenic (FGF-1, FGF-2, KGF, angiopoietin, uPA, VEGF) and antiangiogenic factors (PAI-1, TIMP-1, TSP-1, CD26) were detected in LSCE. In addition, some angiogenic factors (PEDF, PTX3, VEGF) possessed neurotrophic functions. Mice treated with LSCE had 50% to 60% higher salivary flow rate than saline-treated mice at 8 and 12 wk post-IR. Saliva lag time measurements also confirmed that LSCE restored SG function. Histologic analyses of parotids and submandibular glands reported comparable numbers of acinar cells, blood vessels, and parasympathetic nerves and cell proliferation rates in sham IR and LSCE-treated mice, though significantly lower in saline-treated mice. An explant culture method can harvest a large number of LSCs from small pieces of labial glands. LSCE showed clinical potential to mitigate IR-injured SGs.
... The acute functional impairment may be selective radiation damage to the plasma membrane of the secretory cells resulting in disruption of muscarinic receptor-stimulated water secretion. It has also been suggested that the acute dysfunction of the salivary glands may be caused by radiation-induced apoptosis of the serous acinar cells, because genetically engineered mouse models with alterations in key components of the apoptotic pathway do not exhibit salivary gland hypofunction following radiation (36)(37)(38). Although saliva flow rate is significantly reduced and water secretion is selectively hampered, some studies have shown no reduction in number of acinar cells early after radiotherapy (39)(40)(41). ...
... Other potential preventive agents that have been studied in animal models include administration of insulin growth factor-1 (IGF-1); keratinocyte growth factor (KGF); adenoviral vectormediated transfer of complementary DNAs encoding the angiogenic proteins, basic fibroblast growth factor (bFGF) (103); tyrosine kinase inhibitors (104,105); and vascular endothelial growth factor. Intravenous injection of IGF-1 aims to suppress apoptosis because it has been suggested that radiation-induced salivary gland dysfunction in mice results from p53-dependent apoptosis (34,36). In addition to effects on the apoptotic response, IGF-1 has been shown to regulate the cell cycle arrest and DNA repair responses following radiation (106,107). ...
Background:
The most manifest long-term consequences of radiation therapy in the head and neck cancer patient are salivary gland hypofunction and a sensation of oral dryness (xerostomia).
Methods:
This critical review addresses the consequences of radiation injury to salivary gland tissue, the clinical management of salivary gland hypofunction and xerostomia, and current and potential strategies to prevent or reduce radiation injury to salivary gland tissue or restore the function of radiation-injured salivary gland tissue.
Results:
Salivary gland hypofunction and xerostomia have severe implications for oral functioning, maintenance of oral and general health, and quality of life. Significant progress has been made to spare salivary gland function chiefly due to advances in radiation techniques. Other strategies have also been developed, e.g., radioprotectors, identification and preservation/expansion of salivary stem cells by stimulation with cholinergic muscarinic agonists, and application of new lubricating or stimulatory agents, surgical transfer of submandibular glands, and acupuncture.
Conclusion:
Many advances to manage salivary gland hypofunction and xerostomia induced by radiation therapy still only offer partial protection since they are often of short duration, lack the protective effects of saliva, or potentially have significant adverse effects. Intensity-modulated radiation therapy (IMRT), and its next step, proton therapy, have the greatest potential as a management strategy for permanently preserving salivary gland function in head and neck cancer patients.Presently, gene transfer to supplement fluid formation and stem cell transfer to increase the regenerative potential in radiation-damaged salivary glands are promising approaches for regaining function and/or regeneration of radiation-damaged salivary gland tissue.
... Similar to previous studies [2,22,23,26,31,37,39], female animals were used in this study. Nine-week-old (body weight, 30-32 g) female ICR mice were purchased from SLC Inc. (Shizuoka, Japan). ...
... We assessed cleaved caspase-3 as an apoptosis marker and Ki67 as a cellular proliferation marker. Previous studies showed an increase in the apoptosis rate at 24 to 48 hr after low-dose irradiation (2 or 5 Gy) [2,22] in FVB mice. In the present study, apoptosis induction was maintained at 63 days after irradiation. ...
Radiation therapy for head and neck cancer frequently causes salivary gland dysfunction. Pilocarpine is a clinically approved and effective drug that induces saliva secretion, thereby keeping the oral mucosa moist and reducing discomfort in patients, but the effect is transient. We expected that this drug also has beneficial long-term effects that maintain the integrity of salivary glands by reducing, for instance, apoptosis. Here, we examined the effects of long-term pilocarpine administration in irradiated mice. The results indicated that long-term pilocarpine administration significantly improved salivary flow in irradiated mice, suggesting the potential beneficial effects of long-term administration. To elucidate the underlying mechanism, we analyzed the histology, apoptosis, and proliferation of acinar cells, and the expression of functional membrane proteins such as transmembrane member 16A, aquaporin-5, and Na-K-Cl cotransporter. Long-term pilocarpine treatment seemed to decrease irradiation-induced apoptosis, although the change was not statistically significant. The present results indicated that long-term administration of pilocarpine has beneficial effects on salivary flow in irradiated mice, and suggested that long-term administration possibly decreases apoptosis in irradiated salivary glands.
... However, artificial cell culture conditions can alter the differentiation status and physiological responses of cells exposed to various conditions, and the results often fail to translate to whole organism models 14,15 . In addition, immortalized cell cultures require modulation of p53 activity, which is critical for the salivary gland response to DNA damage 16,17 . ...
... Wash slices for 5 min in deionized water. 16. Counterstain slices with DAPI (1µg/mL) for 20 min at RT. 17. Wash slices (once) for 5 min in deionized water. ...
... P2X7R activation also leads to the release of eicosanoids, cytokines, and exosomes and frequently results in cell apoptosis (1,29), including in salivary epithelial cells (10,32). Apoptosis of salivary epithelial cells is a well-described mechanism of ␥-radiation-induced salivary gland dysfunction (4,15,18). ...
... To test a potential therapeutic approach to prevent ␥-radiation-induced salivary gland damage, we determined that a single injection of the P2X7R antagonist A438079 (70 mg/kg) preserved salivary function through day 3 postradiation (Fig. 5C), but multiple injections of A438079 (pre-and postradiation) maintained function long term (30 days, Fig. 5D). Interestingly, the increase in salivary flow seen in irradiated P2X7R Ϫ/Ϫ versus wild-type mice does not correlate with a reduction in cell apoptosis (Fig. 2, B-D), which is in contrast to other mouse models of radiation-induced salivary gland dysfunction (4,14,15,18). This suggests that the P2X7R plays a role in the loss of saliva secretion independent or downstream of apoptosis. ...
Head and neck cancer treatments typically involve a combination of surgery and radiotherapy, often leading to collateral damage to nearby tissues causing unwanted side effects. Radiation damage to salivary glands frequently leads to irreversible dysfunction by poorly understood mechanisms. The P2X7 receptor (P2X7R) is a ligand-gated ion channel activated by extracellular ATP released from damaged cells as "danger signals." P2X7R activation initiates apoptosis and is involved in numerous inflammatory disorders. In this study, we utilized P2X7R knockout (P2X7R-/-) mice to determine the role of the receptor in radiation-induced salivary gland damage. Results indicate a dose-dependent increase in γ-radiation-induced ATP release from primary parotid gland cells of wild-type but not P2X7R-/- mice. Despite these differences, apoptosis levels are similar in parotid glands of wild-type and P2X7R-/- mice 24-72 h after radiation. However, γ-radiation caused elevated prostaglandin E2 (PGE2) release from primary parotid cells of wild-type but not P2X7R-/- mice. To attempt to uncover the mechanism underlying differential PGE2 release, we evaluated the expression and activities of cyclooxygenase and PGE synthase isoforms. There were no consistent trends in these mediators following radiation that could explain the reduction in PGE2 release in P2X7R-/- mice. Irradiated P2X7R-/- mice have stimulated salivary flow rates similar to unirradiated controls, whereas irradiated wild-type mice have significantly decreased salivary flow rates compared with unirradiated controls. Notably, treatment with the P2X7R antagonist A438079 preserves stimulated salivary flow rates in wild-type mice following γ-radiation. These data suggest that P2X7R antagonism is a promising approach for preventing γ-radiation-induced hyposalivation.
... Apoptosis of neutrophils signals the tissue to suppress the inflammatory phase by downregulating the expression of pro-inflammatory cytokines and increasing expression of anti-inflammatory cytokines, thereby facilitating the transition between the inflammatory and the proliferative phase of wound healing [63,64]. Neutrophils in the parotid gland appear to infiltrate later than anticipated, and do not seem to be undergoing apoptosis as expected; we expected to see an increase in PI+ neutrophils at day 2 after IR due to apoptosis of acinar cells occurring 24-48 hours after IR [65]. Proliferation is an important component of the wound healing response; however, compensatory proliferation of the acinar compartment of parotid glands is a persistent phenotype that begins at day 5 after IR and continues through day 90 [35]. ...
Head and neck cancer treatment often consists of surgical resection of the tumor followed by ionizing radiation (IR), which can damage surrounding tissues and cause adverse side effects. The underlying mechanisms of radiation-induced salivary gland dysfunction are not fully understood, and treatment options are scarce and ineffective. The wound healing process is a necessary response to tissue injury, and broadly consists of inflammatory, proliferative, and redifferentiation phases with immune cells playing key roles in all three phases. In this study, select immune cells were phenotyped and quantified, and certain cytokine and chemokine concentrations were measured in mouse parotid glands after IR. Further, we used a model where glandular function is restored to assess the immune phenotype in a regenerative response. These data suggest that irradiated parotid tissue does not progress through a typical inflammatory response observed in wounds that heal. Specifically, total immune cells (CD45+) decrease at days 2 and 5 following IR, macrophages (F4/80+CD11b+) decrease at day 2 and 5 and increase at day 30, while neutrophils (Ly6G+CD11b+) significantly increase at day 30 following IR. Additionally, radiation treatment reduces CD3- cells at all time points, significantly increases CD3+/CD4+CD8+ double positive cells, and significantly reduces CD3+/CD4-CD8- double negative cells at day 30 after IR. Previous data indicate that post-IR treatment with IGF-1 restores salivary gland function at day 30, and IGF-1 injections attenuate the increase in macrophages, neutrophils, and CD4+CD8+ T cells observed at day 30 following IR. Taken together, these data indicate that parotid salivary tissue exhibits a dysregulated immune response following radiation treatment which may contribute to chronic loss of function phenotype in head and neck cancer survivors.
... Among the changes caused by irradiation-induced senescence, DNA damage induces aging and can cause apoptosis 22 . Platelet derivatives have been reported to reduce irradiation-induced apoptosis 23 . ...
Hyposalivation is a common complaint among the elderly, but no established treatment prevents age-induced hyposalivation. Platelet derivatives such as platelet-rich plasma (PRP), platelet-rich fibrin (PRF), and plasma rich in growth factor (PRGF), are used widely in different areas of regenerative medicine to enhance the wound healing processes. This study examined whether the local injection of the supernatant of activated PRP (saPRP) into the salivary gland (SG) could help prevent aging-induced SG dysfunction and explored the mechanisms responsible for the protective effects on the SG hypofunction. The platelets were separated from the blood of male SD rats (220 ± 20 g). saPRP was manufactured by removing the fibrin clot after activating platelet with calcium ionophore 10 μM (A23187). The total protein and TGF-β1 levels were significantly higher in saPRP than in PRP. Human salivary gland epithelial cell(hSGEC) was treated with saPRP or PRP after senescence through irradiation. The significant proliferation of hSGEC was observed in saPRP treated group compared to irradiation only group and irradiation + PRP group. Cellular senescence, apoptosis, and inflammation significantly reduced in saPRP group. The SG function and structural tissue remodeling by the saPRP were investigated with naturally aged mice. The mice were divided into three groups: 3 months old (3 M), 22 months old (22 M), and 22 months old treated with saPRP (22 M + saPRP). Salivary flow rate and lag time were significantly improved in 22 M + saPRP group compared to 22 M group. The histologic examinations showed the significant proliferation of acinar cell in 22 M + saPRP group. The decrease of senescence, apoptosis, and inflammation observed by western blot in 22 M + saPRP group. The saPRP induced the proliferation of hSGECs, leading to a significant decrease in cellular senescence via decrease inflammation and apoptosis, in vitro. Moreover, the acini cells of the salivary gland were regenerated, and the salivary function increased in aged mice. These results showed that saPRP could be a treatment agent against aging-induced SG dysfunction.
... 4 Mice experience a significant reduction in acinar cells, salivary flow rate, and changes in saliva composition shortly after IR exposure. [69][70][71] Reports have noted that chronic hyposalivation begins between 30 and 300 days following IR, with fibrosis occurring as early as 30 days after receiving fractionated IR in minipigs and over 4 months in rodents. 72,73 In addition to immunostaining of SG-gland biopsies showing fibrotic lesions, genomic profiling has linked increased collagen deposition in irradiated SGs to intraglandular up-regulation of fibrosis pathways. ...
Salivary gland (SG) extracellular matrix (ECM) has a major influence on tissue development, homeostasis, and tissue regeneration after injury. During aging, disease, and physical insult, normal remodeling of the SG microenvironment (i.e. ECM) becomes dysregulated, leading to alterations in matrix composition which disrupt tissue architecture/structure, alter cell activity, and negatively impact gland function. Matrix metalloproteinases (MMPs) are a large and diverse family of metalloendopeptidases which play a major role in matrix degradation and are intimately involved in regulating development and cell function; dysregulation of these enzymes leads to the production of a fibrotic matrix. In the SG this altered fibrotic ECM (or cell microenvironment) negatively impacts normal cell function and the effectiveness of gene and stem cell therapies which serve as a foundation for many SG regenerative therapies. For this reason, prospective regenerative strategies should prioritize the maintenance and/or restoration of a healthy SG ECM. Mesenchymal stem cells (MSCs) have great potential for mitigating damage to the SG microenvironment by ameliorating inflammation, reducing fibrosis, and repairing the damaged milieu of extracellular regulatory cues, including the matrix. This review addresses our current understanding of the impact of aging and disease on the SG microenvironment and suggests critical deficiencies and opportunities in ECM-targeted therapeutic interventions.
... 7,19,46 One of the hypotheses regards the action of ionizing radiation to interrupt the transduction of signals in the membranes of the acini. 47 In animal studies, some of the phenomena observed included an increase in the number of apoptotic cells, 48 a reduction in the expression of aquaporins, 49 and hypo vascularization. 50 Other effects were fibrous tissue formation, cytoplasmic vacuolization, edema, 51 and up to a 30% reduction in the original glandular volume. ...
Objective(s):
This scoping review aims to map the scientific literature on the therapies currently available for physical salivary stimulation in individuals with hyposalivation caused by radiotherapy.
Study design:
Studies were included when they comprised the target population of adult individuals receiving radiotherapy of the head and neck region and who developed or were at risk of developing hyposalivation. Two reviewers selected the studies and extracted data on the type of physical salivary stimulation therapy used, the degree of glandular tissue involvement, and the percentage of salivary flow alteration. Therapies were classified according to either prophylactic application (before/during radiotherapy) or therapeutic application (post-radiotherapy).
Results:
Sixteen articles were included: 4 tested transcutaneous electrical nerve stimulation (TENS), 3 studied low-level lasers, 7 researched acupunctures, and 2 investigated acupuncture-like TENS. The outcomes of the prophylactic studies indicated beneficial effects (similar salivary flow or reduced salivary flow loss), although most studies did not include a comparable control group. Therapeutic studies presented conflicting results.
Conclusion(s):
Prophylactic therapies of physical salivary stimulation may produce better effects than therapeutic applications. However, the protocols best indicated could not be defined. Well-designed, controlled clinical trials should be researched in the future to support the clinical recommendation of any of these treatments.
... More speci cally, radiotherapy for head and neck cancer reduces SG stem cell numbers and, therefore SG cell numbers. Thus, restoring SG numbers using stem cells has been suggested as a potential treatment for radiotherapy-induced SG cell loss [3][4][5] . ...
Radiation therapy for head and neck cancer damages local stem cells and epithelial cells in salivary glands (SG), leading to irreversible SG dysfunction. Biological understanding of the responses of tissue-resident stem cells to gravity is required to develop therapeutic strategies for damaged tissue regeneration. In this study, we successfully isolated human minor salivary gland stem cells (huMSGSCs), which have high proliferation rates, express multiple stem markers, and can be differentiated into mesenchymal cell types. Cell proliferation, sphere-forming ability, stemness marker expressions, and epithelial differentiation potentials were checked after exposing huMSGSCs short-term to hypergravity (HyperG) or microgravity (MicroG). Proliferation after exposure to HyperG (40 G ) was greater than exposure to 1G, but no difference was observed between MicroG (10 − 3 G) and 1G. Numbers of large spheres were significantly higher post-HyperG and lower post-MicroG than at 1G, whereas numbers of small spheres were significantly lower post-HyperG and higher post-MicroG. The expressions of stemness markers (CD90, LGR5, CD29, and CD24) and junction markers (ZO-1 and ZO-2) were increased post-HyperG. Furthermore, increases in PAS staining and the gene expressions of albumin and CK19 confirmed that HyperG enhances the epithelial differentiation potential of huMSGSCs. This study shows that cultivating huMSGSCs under HyperG conditions enhances stemness and that a gravity control system could be applied to modulate huMSGSC functions.
... IR-induced dry mouth is related to apoptosis and the senescence of salivary gland cells in both mouse and pig models [16][17][18][19][20]. TUNEL staining of SMGs collected 24 h and 7 days after IR indicated that the number of apoptotic cells significantly increased after IR, as expected, which was significantly repressed by S1P pretreatment (Figures 2A,B and S4). ...
Radiotherapy for head-and-neck cancers frequently causes long-term hypofunction of salivary glands that severely compromises quality of life and is difficult to treat. Here, we studied effects and mechanisms of Sphingosine-1-phosphate (S1P), a versatile signaling sphingolipid, in preventing irreversible dry mouth caused by radiotherapy. Mouse submandibular glands (SMGs) were irradiated with or without intra-SMG S1P pretreatment. The saliva flow rate was measured following pilocarpine stimulation. The expression of genes related to S1P signaling and radiation damage was examined by flow cytometry, immunohistochemistry, quantitative RT-PCR, Western blotting, and/or single-cell RNA-sequencing. S1P pretreatment ameliorated irradiation-induced salivary dysfunction in mice through a decrease in irradiation-induced oxidative stress and consequent apoptosis and cellular senescence, which is related to the enhancement of Nrf2-regulated anti-oxidative response. In mouse SMGs, endothelial cells and resident macrophages are the major cells capable of producing S1P and expressing the pro-regenerative S1P receptor S1pr1. Both mouse SMGs and human endothelial cells are protected from irradiation damage by S1P pretreatment, likely through the S1pr1/Akt/eNOS axis. Moreover, intra-SMG-injected S1P did not affect the growth and radiosensitivity of head-and-neck cancer in a mouse model. These data indicate that S1P signaling pathway is a promising target for alleviating irradiation-induced salivary gland hypofunction.
... Furthermore, an acinar abnormality was thought to be related to radiationinduced downregulation of the aquaporin-5 present in the apical plasma membrane of the acinar cells (Li et al. 2006). The histopathological changes in the salivary glands following irradiation may be attributed to radiation-induced apoptosis mediated by p53 activation (Avila et al. 2009). The detected nuclear abnormalities, that is, pleomorphism and hyperchromatism in the nuclei of irradiated SMGs and partial cell damage were supported by the findings of another study which suggested that the secretory granules of acinar cells are damaged by radiation-induced lipid peroxidation, leading to the lysis of these cells and serous acinar cells containing heavy metals, including zinc, iron, and copper ions in the secretory granules that increase the sensitivity of the salivary glands to ionizing radiations (IRs) (Krishnan et al. 2017). ...
Background & purpose:
Salivary gland damage remains a problem despite advances in radiotherapy schedules for head and neck cancer. Kaempferol, a natural flavonoid, found in several fruits and vegetables, is a good antioxidant. This study was designed to evaluate the possible protective effects of kaempferol on submandibular glands (SMGs) of rats exposed to fractionated gamma irradiation.
Material and methods:
Twenty-four male adult Wistar albino rats were included in this study and assigned to three groups (n= 8). Rats in group K received kaempferol orally in five doses at a dose of 10 mg/kg/2days for 10 days. Meanwhile, rats in group R were subjected to fractionated whole-body gamma irradiation at a dose of 2 Gy/5 days/week for 2 weeks (20 Gy), and the KR group received kaempferol as group K and then was subjected to a fractionated whole- body gamma irradiation as group R. SMG samples were collected on days 1 and 7 after the last radiation session; and processed for histopathological and immunohistochemical investigations.
Results:
The SMGs of group R showed focal atrophy and degeneration. Acini showed vacuolization and had pyknotic hyperchromatic nuclei. Striated ducts degenerated, shrunken, and were surrounded by empty spaces. The percentage of areas covered by cyclooxygenase 2 (COX-2) significantly increased, whereas the percentage of areas covered by proliferating cell nuclear antigen (PCNA) significantly decreased compared with those in group K. Cotreatment with kaempferol (group KR) partially preserved normal gland architecture where acinar vacuolation and degeneration were almost absent; however, some ducts degenerated. A significant decrease in the percentage of areas covered by COX-2 and a significant increase in the percentage of areas covered by PCNA was observed compared with those in group R.
Conclusion:
Kaempferol has a possible radioprotective effect on the SMGs of rats exposed to fractionated gamma irradiation.
... In a study, the accumulation of the p53 protein due to radiation was found to activate the up-regulated modulator of apoptosis (PUMA) and Bcl-2-associated X protein (Bax) target genes with a pro-apoptotic effect. 24 The radiotherapy treatment areas are typically within the range of the salivary glands. Thus, patient develops hyposalivation and xerostomia. ...
... Radiation therapy forms an integral component of the multimodal management of the disease. (1) Salivary glands are sensitive to radiation. The parotid gland is more radiosensitive when compared to the submandibular gland. ...
Introduction: Radiation therapy forms an integral component in the management of head and neck cancers. The parotid gland is
radiosensitive to irradiation which leads to xerostomia and its subsequent harmful effects on patient health. Curcumin extract is one of the
most popular natural free radical scavengers and antioxidant; which has ameliorating effect on many organs during radiotherapy.
Aim of the work: This study investigated the effect of curcumin extract on parotid gland during radiotherapy.
Methods: 50 adult albino rats were selected for the study and divided into control group (5rats) and experimental group (45 rats) which
was subdivided into subgroup, and (15 rats each). The control group had neither irradiation nor a supplementation. The experimental
group was irradiated with a single dose of 15 GY .Subgroup : did not receive any supplementation but only irradiated Subgroup:
supplemented with curcumin 200mg/kg orally for 15 days followed by irradiation. Subgroup: supplemented with curcumin 200mg/kg
orally for 15 day followed by irradiation and the curcumin supplementation extended for two weeks after the irradiation day. Five animals
of each experimental subgroup were sacrificed at 1, 7 and 14 days from the irradiation day (subgroup III, from the last irradiation), the
parotid glands were excised and prepared for histological, immunohistochemical, comet assay and malondialdhyde studies.
Results: curcumin extract had ameliorating effect on the acinar destruction caused by irradiation, it enhanced acinar cell proliferation to
compensate the cell loss, decreased the DNA destruction and lipid peroxidation formed as a result of irradiation
... We have reported that in radiation-induced cellular senescence in pig parotid glands, treatment with transient Hedgehog activation after radiation effectively inhibited cellular senescence and preserved the proliferation capacities of pig parotid cells [23]. P53 signaling mediates the cellular senescence and apoptosis caused by radiation in various tissues, including mouse salivary glands [7,25]. Therefore, we examined the levels of P53 protein and mRNA in pig parotids using Western blotting and qRT-PCR. ...
Salivary gland function is commonly and irreversibly damaged by radiation therapy for head and neck cancer. This damage greatly decreases the patient’s quality of life and is difficult to remedy. Previously, we found that the transient activation of Hedgehog signaling alleviated salivary hypofunction after radiation in both mouse and pig models through the inhibition of radiation-induced cellular senescence that is mediated by resident macrophages in mouse submandibular glands. Here we report that in swine parotid glands sharing many features with humans, the Hedgehog receptor PTCH1 is mainly expressed in macrophages, and levels of PTCH1 and multiple macrophage markers are significantly decreased by radiation but recovered by transient Hedgehog activation. These parotid macrophages mainly express the M2 macrophage marker ARG1, while radiation promotes expression of pro-inflammatory cytokine that is reversed by transient Hedgehog activation. Hedgehog activation likely preserves parotid macrophages after radiation through inhibition of P53 signaling and consequent cellular senescence. Consistently, VEGF, an essential anti-senescence cytokine downstream of Hedgehog signaling, is significantly decreased by radiation but recovered by transient Hedgehog activation. These findings indicate that in the clinically-relevant swine model, transient Hedgehog activation restores the function of irradiated salivary glands through the recovery of resident macrophages and the consequent inhibition of cellular senescence and inflammation.
... First, it was discovered that the necessary condition for IR to cause the apoptosis of SGs was the expression of p53 in the body. P53 is a tumor suppressor that is closely related to the regulation of DNA damage repair, cell cycle arrest, and cell apoptosis [59]. ...
Radiation therapy is a common treatment for head and neck cancers. However, because of the presence of nerve structures (brain stem, spinal cord, and brachial plexus), salivary glands (SGs), mucous membranes, and swallowing muscles in the head and neck regions, radiotherapy inevitably causes damage to these normal tissues. Among them, SG injury is a serious adverse event, and its clinical manifestations include changes in taste, difficulty chewing and swallowing, oral infections, and dental caries. These clinical symptoms seriously reduce a patient’s quality of life. Therefore, it is important to clarify the mechanism of SG injury caused by radiotherapy. Although the mechanism of radiation-induced SG injury has not yet been determined, recent studies have shown that the mechanisms of calcium signaling, microvascular injury, cellular senescence, and apoptosis are closely related to oxidative stress. In this article, we review the mechanism by which radiotherapy causes oxidative stress and damages the SGs. In addition, we discuss effective methods to prevent and treat radiation-induced SG damage.
... Cellular senescence is a pathophysiological process that arrests cell growth and inhibits cell proliferation, exhibiting both beneficial (e.g., tissue repair and tumor suppression) and detrimental (e.g., tumorigenesis and aging) outcomes. Previous research demonstrated that apoptotic events lead to SG hypofunction after IR [12], and recent reports indicated that IR-induced SG dysfunction results from lasting damage caused by senescence to salivary stem cell niches [13]. The debate over the role of IR-induced cell apoptosis in SG hypofunction may originate from experimental factors, such as the radiation dose, delivery method (e.g., targeted vs. whole head IR), analytical time point post-IR, and tolerance of species and various mouse models to IR [14]. ...
Small extracellular vesicles (sEV) facilitate signaling molecule transfer among cells. We examined the therapeutic efficacy of human dental pulp stem cell-derived sEV (hDPSC-sEV) against cellular senescence in an irradiated-submandibular gland mouse model. Seven-week-old mice were exposed to 25 Gy radiation and randomly assigned to control, phosphate-buffered saline (PBS), or hDPSC-sEV groups. At 18 days post-irradiation, saliva production was measured; histological and reverse transcription-quantitative PCR analyses of the submandibular glands were performed. The salivary flow rate did not differ significantly between the PBS and hDPSC-sEV groups. AQP5-expressing acinar cell numbers and AQP5 expression levels in the submandibular glands were higher in the hDPSC-sEV group than in the other groups. Furthermore, compared with non-irradiated mice, mice in the 25 Gy + PBS group showed a high senescence-associated-β-galactosidase-positive cell number and upregulated senescence-related gene (p16INK4a, p19Arf, p21) and senescence-associated secretory phenotypic factor (MMP3, IL-6, PAI-1, NF-κB, and TGF-β) expression, all of which were downregulated in the hDPSC-sEV group. Superoxide dismutase levels were lower in the PBS group than in the hDPSC-sEV group. In summary, hDPSC-sEV reduced inflammatory cytokine and senescence-related gene expression and reversed oxidative stress in submandibular cells, thereby preventing irradiation-induced cellular senescence. Based on these results, we hope to contribute to the development of innovative treatment methods for salivary gland dysfunction that develops after radiotherapy for head and neck cancer.
... Saliva secretion occurs in acinar epithelial cells and is dependent upon a rich blood supply by microvessels surrounding acini. IR causes the irreversible impairment of acinar epithelial cells and microvascular endothelial cells in SGs through multiple mechanisms (3)(4)(5). Among the three pairs of major SGs, submandibular glands (SMG) produce two thirds of the total volume of the whole saliva in the unstimulated state and approximately 46% of that after stimulation (6), and are the focus of many research studies including ours. ...
Irreversible hypofunction of salivary glands is a common side effect of radiotherapy for head and neck cancer and is difficult to remedy. Recent studies indicate that transient activation of Hedgehog signaling rescues irradiation-impaired salivary function in animal models, but the underlying mechanisms are largely unclear. Here, we show in mice that activation of canonical Gli-dependent Hedgehog signaling by Gli1 gene transfer is sufficient to recover salivary function impaired by irradiation. Salivary gland cells responsive to Hedgehog/Gli signaling comprised small subsets of macrophages, epithelial cells, and endothelial cells, and their progeny remained relatively rare long after irradiation and transient Hedgehog activation. Quantities and activities of salivary gland resident macrophages were substantially and rapidly impaired by irradiation and restored by Hedgehog activation. Conversely, depletion of salivary gland macrophages by clodronate liposomes compromised the restoration of irradiation-impaired salivary function by transient Hedgehog activation. Single-cell RNA sequencing and qRT-PCR of sorted cells indicated that Hedgehog activation greatly enhances paracrine interactions between salivary gland resident macrophages, epithelial progenitors, and endothelial cells through Csf1, Hgf, and C1q signaling pathways. Consistently, expression of these paracrine factors and their receptors in salivary glands decreased following irradiation but were restored by transient Hedgehog activation. These findings reveal that resident macrophages and their prorepair paracrine factors are essential for the rescue of irradiation-impaired salivary function by transient Hedgehog activation and are promising therapeutic targets of radiotherapy-induced irreversible dry mouth.
Significance: These findings illuminate a novel direction for developing effective treatment of irreversible dry mouth, which is common after radiotherapy for head and neck cancer and for which no effective treatments are available.
Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/80/24/5531/F1.large.jpg.
See related commentary by Coppes, p. 5462
... However, activation of p53 transcription induced by DNA damage appears to play a key role in acinar cell radiosensitivity. The accumulation of the p53 protein activates the up-regulated modulator of apoptosis (PUMA) and Bcl-2-associated X protein (Bax) target genes (Avila et al., 2009;Grundmann et al., 2009). These genes have a pro-apoptotic effect, resulting in a decrease in the number of acinar cells in a dose-dependent relationship (Cheng et al., 2011;Acauan et al., 2015). ...
Introduction
Head and neck radiotherapy can cause hypofunction of the salivary glands. Many studies report that laser photobiomodulation (PBM) is able to minimize radiation-induced hyposalivation, yet there is no consensus about its effects.
Objective
To carry out a meta-analysis of controlled clinical trials that used PBM to prevent radiation-induced hyposalivation. Methods: A systematic search was performed through Embase, Medline/PubMed, Cochrane, EBSCO, Scopus, LILACS and Web of Science databases. The strategy included comparisons of the effect of PBM with placebo/clinical follow-up on unstimulated and/or stimulated salivary flow in patients undergoing head and neck radiotherapy.
Results
Six clinical trials were included, five of which were used for meta-analysis. Evidence was observed between the use of PBM and increased unstimulated salivary flow (MD 0.20 mL/min, 95 % Cl 0.10−0.30, I² = 96 %, p < 0.00001) and in stimulated salivary flow (MD 0.27 mL/min, 95 % CI 0.08−0.46, I² = 95 %, p < 0.00001).
Conclusion
PBM appears to minimize radiation-induced hyposalivation.
... A study protocol has been published recently to evaluate the effect of PBM in patients with antihypertensive drug-induced xerostomia (Varellis et al., 2020) so that may endorse the need for further studies that deal with this population. Our review also did not retrieve studies on patients who had undergone radiotherapy, despite the recognized glandular changes associated with the irradiation (Avila et al., 2009) that involved xerostomia/hyposalivation, both dose-dependent. This might be explained because one of our exclusion criteria was acute complaints, where PBM therapy may have direct exposure or treatment areas close of the tumour site (Elad et al., 2018), as cancers cells could be exposed to laser irradiation (Hamblin et al., 2018). ...
The objective was to explore the effectiveness of photobiomodulation therapy for treating patients who suffer xerostomia and/or hyposalivation due to the most prevalent clinical diagnoses. We searched PubMed, Scopus, Web of Science, CINAHL, and Cochrane Library for randomised or clinical controlled trials published until 31 October 2019. Risk of bias assessment and meta-analysis were conducted using the Cochrane tools. A total of 274 records were retrieved and 11 met the inclusion criteria. Interventions whose parameters ranged between wavelengths of 790-830nm (infrared), 30-120mW of power, and an energy density below 30J/cm-2 were associated with improvements in xerostomia/hyposalivation. As for the assessment of methodological quality, 10 of the 11 articles included had a high risk of overall bias. Only 3 articles provided sufficient information to conduct a meta-analysis for quality of life, compared with placebo in patients with burning mouth syndrome, showing a standardised mean difference between groups from baseline of -0.90 (-1.48;-0.32). The present review and meta-analysis suggest that photobiomodulation therapy is an effective, non-invasive and safe approach in patients with xerostomia. However, despite the potential, it is not possible to reach a reliable consensus on the parameters to be used, and future studies should be conducted by standardising intervention protocols.
... The dual roles of JNK signaling as a mediator of apoptosis or as a mediator of cell proliferation raises the question of how one of these downstream outcomes become favored in a temporal manner following radiation damage. Some insights to this question can be gleaned from previous work on the apoptotic response of irradiated salivary glands, whereby apoptosis can be detected as early as four hours after radiation treatment, peaks at 24 hours, and returns to basal levels by 72 hours [41][42][43][44]. In contrast, the current study demonstrates that radiationinduced JNK signaling is observed at day 5 post-radiation, a time point beyond the apoptotic response and the beginning of the compensatory proliferation response. ...
Radiotherapy is a common treatment option for head and neck cancer patients; however, the surrounding healthy salivary glands are often incidentally irradiated during the process. As a result, patients often experience persistent xerostomia and hyposalivation, which deceases their quality of life. Clinically, there is currently no standard of care available to restore salivary function. Repair of epithelial wounds involves cellular proliferation and establishment of polarity in order to regenerate the tissue. This process is partially mediated by protein kinase C zeta (PKCζ), an apical polarity regulator; however, its role following radiation damage is not completely understood. Using an in vivo radiation model, we show a significant decrease in active PKCζ in irradiated murine parotid glands, which correlates with increased proliferation that is sustained through 30 days post-irradiation. Additionally, salivary glands in PKCζ null mice show increased basal proliferation which radiation treatment did not further potentiate. Radiation damage also activates Jun N-terminal kinase (JNK), a proliferation-inducing mitogen-activated protein kinase normally inhibited by PKCζ. In both a PKCζ null mouse model and in primary salivary gland cell cultures treated with a PKCζ inhibitor, there was increased JNK activity and production of downstream proliferative transcripts. Collectively, these findings provide a potential molecular link by which PKCζ suppression following radiation damage promotes JNK activation and radiation-induced compensatory proliferation in the salivary gland.
... Interestingly, many of these cells are found in the acinar compartment of the gland, which has been shown to replenish itself during homeostasis and is thought to contribute to gland regeneration 10 . Label retaining cells are present for at least 30 days post-radiation 9 , but function of the salivary glands is not restored 7,11,12 . Understanding the mechanisms that prevent LRCs from repairing irradiated salivary glands will be instrumental in designing therapies to target specific populations of endogenous cells to promote wound healing without the need for transplantation. ...
Xerostomia and salivary hypofunction often result as a consequence of radiation therapy for head and neck cancers, which are diagnosed in roughly 60,000 individuals every year in the U.S. Due to the lack of effective treatments for radiation-induced salivary hypofunction, stem cell-based therapies have been suggested to regenerate the irradiated salivary glands. Pharmacologically, restoration of salivary gland function has been accomplished in mice by administering IGF-1 shortly after radiation treatment, but it is not known if salivary stem and progenitor cells play a role. We show that radiation inactivates aPKCζ and promotes nuclear redistribution of Yap in a population of label-retaining cells in the acinar compartment of the parotid gland (PG)- which comprises a heterogeneous pool of salivary progenitors. Administration of IGF-1 post-radiation maintains activation of aPKCζ and partially rescues Yap's cellular localization in label retaining cells, while restoring salivary function. Finally, IGF-1 fails to restore saliva production in mice lacking aPKCζ, demonstrating the importance of the kinase as a potential therapeutic target.
Objectives
Salivary gland injury is one of the most common complications of radiotherapy in head‐and‐neck cancers. This study investigated the mechanism by which rapamycin prevents irradiation (IR)‐induced injury in the parotid glands.
Materials and Methods
Miniature pigs either received (a) no treatment (NT), (b) IR in the right parotid gland for 5 consecutive days (IR), or intraperitoneal administration of rapamycin (Rap) 1 h prior to IR (IR + Rap). Tissues were collected at three distinct time points (24 h, 4 weeks, and 16 weeks) after IR. Histological analyses, western blot, and real‐time reverse transcriptase‐polymerase chain reaction were performed to explore the mechanisms of IR‐induced injury in the parotid gland.
Results
Rapamycin treatment maintained parotid salivary flow 16 weeks post‐IR, preserved the number of acinar cells, and reduced parotid tissue fibrosis, as well as reduced apoptosis levels, decreased cleaved caspase‐3 expression, and increased the Bcl‐2/Bax ratio in the parotid glands. Autophagy marker LC3B was upregulated by rapamycin after IR, while P62 expression was downregulated. Rapamycin reduced the expression of pro‐inflammatory factors and the mesenchymal tissue fibrosis following IR.
Conclusions
Rapamycin maintains gland homeostasis after IR by decreasing apoptosis, reducing the expression of pro‐inflammatory factors, and enhancing autophagy.
Salivary gland damage and hypofunction result from various disorders, including autoimmune Sjögren's disease (SjD) and IgG4-related disease (IgG4-RD), as well as a side effect of radiotherapy for treating head and neck cancers. There are no therapeutic strategies to prevent the loss of salivary gland function in these disorders nor facilitate functional salivary gland regeneration. However, ongoing aquaporin-1 gene therapy trials to restore saliva flow show promise. To identify and develop novel therapeutic targets, we must better understand the cell-specific signaling processes involved in salivary gland regeneration. Transforming growth factor-β (TGF-β) signaling is essential to tissue fibrosis, a major endpoint in salivary gland degeneration, which develops in the salivary glands of patients with SjD, IgG4-RD, and radiation-induced damage. Though the deposition and remodeling of extracellular matrix proteins are essential to repair salivary gland damage, pathological fibrosis results in tissue hardening and chronic salivary gland dysfunction orchestrated by multiple cell types, including fibroblasts, myofibroblasts, endothelial cells, stromal cells, and lymphocytes, macrophages, and other immune cell populations. This review is focused on the role of TGF-β signaling in the development of salivary gland fibrosis and the potential for targeting TGF-β as a novel therapeutic approach to regenerate functional salivary glands. The studies presented highlight the divergent roles of TGF-β signaling in salivary gland development and dysfunction and illuminate specific cell populations in damaged or diseased salivary glands that mediate the effects of TGF-β. Overall, these studies strongly support the premise that blocking TGF-β signaling holds promise for the regeneration of functional salivary glands.
Salivary glands are indirectly damaged during radiotherapy for head and neck cancer, resulting in acute and chronic hyposalivation. Current treatments for radiation-induced hyposalivation do not permanently restore function to the gland; therefore, more mechanistic understanding of the damage response is needed to identify therapeutic targets for lasting restoration. Energy metabolism reprogramming has been observed in cancer and wound healing models to provide necessary fuel for cell proliferation; however, there is limited understanding of alterations in energy metabolism reprogramming in tissues that fail to heal. We measured extracellular acidification and oxygen consumption rates, assessed mitochondrial DNA copy number, and tested fuel dependency of irradiated primary salivary acinar cells. Radiation treatment leads to increases in glycolytic flux, oxidative phosphorylation, and ATP production rate at acute and intermediate time points. In contrast, at chronic radiation time points there is a significant decrease in glycolytic flux, oxidative phosphorylation, and ATP production rate. Irradiated salivary glands exhibit significant decreases in spare respiratory capacity and increases in mitochondrial DNA copy number at days 5 and 30 post-treatment, suggesting a mitochondrial dysfunction phenotype. These results elucidate kinetic changes in energy metabolism reprogramming of irradiated salivary glands that may underscore the chronic loss of function phenotype.
Head and neck cancer treatment often consists of surgical resection of the tumor followed by ionizing radiation (IR), which can damage surrounding tissues and cause adverse side effects. The underlying mechanisms of radiation-induced salivary gland dysfunction are not fully understood, and treatment options are scarce and ineffective. The wound healing process is a necessary response to tissue injury, and broadly consists of inflammatory, proliferative, and redifferentiation phases with immune cells playing key roles in all three phases. In this study, select immune cells were phenotyped and quantified, and certain cytokine and chemokine concentrations were measured in mouse parotid glands after IR. Further, we used a model where glandular function is restored to assess the immune phenotype in a regenerative response. These data suggest that irradiated parotid tissue does not progress through a typical inflammatory response observed in wounds that heal. Specifically, total immune cells (CD45+) decrease at days 2 and 5 following IR, macrophages (F4/80+CD11b+) decrease at day 2 and 5 and increase at day 30, while neutrophils (Ly6G+CD11b+) significantly increase at day 30 following IR. Additionally, radiation treatment reduces CD3- cells at all time points, significantly increases CD3+/CD4+CD8+ double positive cells, and significantly reduces CD3+/CD4-CD8- double negative cells at day 30 after IR. Previous data indicate that post-IR treatment with IGF-1 restores salivary gland function at day 30, and IGF-1 injections attenuate the increase in macrophages, neutrophils, and CD4+CD8+ T cells observed at day 30 following IR. Taken together, these data indicate that parotid salivary tissue exhibits a dysregulated immune response following radiation treatment which may contribute to chronic loss of function phenotype in head and neck cancer survivors.
Salivary gland (SG) dysfunction, due to radiotherapy, disease, or aging, is a clinical manifestation that has the potential to cause severe oral and/or systemic diseases and compromise quality of life. Currently, the standard-of-care for this condition remains palliative. A variety of approaches have been employed to restore saliva production, but they have largely failed due to damage to both secretory cells and the extracellular matrix (niche). Transplantation of allogeneic cells from healthy donors has been suggested as a potential solution, but no definitive population of SG stem cells, capable of regenerating the gland, has been identified. Alternatively, mesenchymal stem cells (MSCs) are abundant, well characterized, and during SG development/homeostasis engage in signaling crosstalk with the SG epithelium. Further, the trans-differentiation potential of these cells and their ability to regenerate SG tissues have been demonstrated. However, recent findings suggest that the “immuno-privileged” status of allogeneic adult MSCs may not reflect their status post-transplantation. In contrast, autologous MSCs can be recovered from healthy tissues and do not present a challenge to the recipient’s immune system. With recent advances in our ability to expand MSCs in vitro on tissue-specific matrices, autologous MSCs may offer a new therapeutic paradigm for restoration of SG function.
Understanding the transcriptional landscape that results in chronic salivary hypofunction after irradiation will help identify injury mechanisms and develop regenerative therapies. We present scRNA-seq analysis from control and irradiated murine parotid glands collected 10 months after irradiation. We identify a population of secretory cells defined by specific expression of Etv1, which may be an acinar cell precursor. Acinar and Etv1+ secretory express Ntrk2 and Erbb3, respectively while the ligands for these receptors are expressed in myoepithelial and stromal cells. Furthermore, our data suggests that secretory cells and CD4+CD8+T-cells are the most transcriptionally affected during chronic injury with radiation, suggesting active immune involvement. Lastly, evaluation of cell-cell communication networks predicts that neurotrophin, neuregulin, ECM, and immune signaling are dysregulated after irradiation, and thus may play a role in the lack of repair. This resource will be helpful to understand cell-specific pathways that may be targeted to repair chronic damage in irradiated glands.
Many patients treated for head and neck cancers experience salivary gland hypofunction due to radiation damage. Understanding the mechanisms of cellular damage induced by radiation treatment is important in order to design methods of radioprotection. In addition, it is crucial to recognize the indirect effects of IR and the systemic responses that may alter saliva secretion. In this study, radiation was delivered to murine submandibular glands (SMG) bilaterally, using a 137Cs gamma ray irradiator, or unilaterally, using a small animal radiation research platform (SARRP). Analysis at 3, 24 and 48 hours showed dynamic changes in mRNA levels and protein abundance in SMG irradiated bilaterally. Unilateral irradiation using the SARRP caused similar changes in the irradiated SMG, as well as significant off-target, bystander effects in the non-irradiated contralateral SMG.
Dry mouth is the main manifestation of Sjögren syndrome (SS). Quercetin has been reported to alleviate radiation‐induced salivary gland damage, yet the effect of quercetin on SS‐caused salivary gland damage remains unclear. This study aimed to investigate the effects of quercetin on SS‐induced salivary gland damage and the mechanism underlying its therapeutic potential in SS. Here, NOD/Ltj mice were used to spontaneously mimic SS‐induced salivary gland inflammation in vivo and salivary gland epithelial cells (SGECs) were stimulated by interferon‐γ (IFN‐γ) to mimic cell inflammation in vitro. Results showed that quercetin significantly reduced loss of saliva flow, salivary gland damage, cell apoptosis, and inflammatory response in NOD/Ltj mice. Quercetin treatment also significantly reduced the increased serum leptin (LP) levels in NOD/Ltj mice. Furthermore, quercetin blocked the increases in the expression of obesity receptor (OB‐R) and its downstream Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling in the salivary glands. In vitro experiments confirmed that quercetin could protect SGECs from IFN‐γ‐induced cell apoptosis and inflammation through the LP/OB‐R‐activated JAK2/STAT3 signaling. Hence, quercetin might protect against SS‐induced salivary gland damage by relieving cell apoptosis and inflammation by inhibiting the LP/OB‐R signaling, providing a new perspective for treating SS‐induced dry mouth.
Photobiomodulation (PBM) is now over 50 years old and has recently started to gain more widespread acceptance within the medical community. Alleviation of the side effects of cancer therapy has so far been one of the most impressive applications of PBM and the focus of this chapter. I will cover the mechanism of action and ask whether the effects of PBM on cancer cells and tumors is more likely to be positive or negative. There are some ways that PBM could actually attack the cancer either directly or indirectly, or in combination with other cytotoxic therapies. Finally, I will summarize the clinical applications of PBM against such side effects as oral mucositis, radiation dermatitis, dysphagia, hyposalivation and xerostomia, taste alterations, trismus, head and neck lymphedema, and voice and speech alterations.
Head and neck cancers commonly require radiation as part of treatment. Irradiated patients suffer from severe salivary gland impairment. In this study, we investigated the role of silver nanoparticles (AgNPs) in preventing radiation-induced changes in Albino rats’ submandibular glands (SMG). Thirty adult male Albino rats were divided into three groups ten rats each; group I (control) and group II (irradiated group (IR)) received a single dose of 15 Gray (Gy), and group III (AgNPs + IR) received 150 µg/kg of AgNPs and a single dose of 15 Gy. Sialochemical analysis of salivary function has been evaluated. Salivary levels of 8 hydroxydeoxyguanosine (8-OHdG), nitric oxide (NO), and total antioxidant capacity (TAC) have been measured. Histological and ultrastructural examinations of the salivary gland have been assessed. Immunohistochemical expression of malondialdehyde (MDA), superoxide dismutase (SOD), and inducible nitric oxide synthetase (iNOS) were detected. The results showed a significant decrease in salivary gland function and total protein levels in the IR group, contrary to group III. The 8-OHdG and NO levels and the immunohistochemical expression of MDA and iNOS were significantly increased in the IR group. However, the levels of TAC and SOD were significantly decreased. IR group showed atrophy of acinar cells with signs of degeneration, wide intercellular spaces, and cytoplasmic vacuolizations in addition to apoptotic cells. The results of group III showed protection of the submandibular salivary gland from radiation. We concluded that silver nanoparticles could have a protective role against radiotherapy through their antioxidant activity and scavenging of reactive oxygen species (ROS).
Over 80% of patients undergoing radiotherapy (RT) for head and neck cancer (HNC) suffer reduced saliva secretion and dry mouth symptoms due to salivary gland damage. Although therapeutic interventions to alleviate such RT-induced damage are available, long-term hypofunction remains a significant issue. Therefore, novel therapeutic solutions to prevent irradiation (IR)-induced salivary gland damage are required. This study explored the protective effect of inorganic nitrate in preventing IR-induced salivary gland injury via pyroptosis suppression, both in vivo and in vitro. In the treatment group, C57BL/6 mice were pretreated with 2 mmol/L NaNO3 supplied in drinking water one week before a single-dose of 15 Gy IR in the submandibular gland (SMG) region. Human vein endothelial cells (HUVECs) and mice SMG cells were treated with 10 or 100 μmol/L NaNO3 2 h before a single-dose of 8 Gy IR. In vivo, IR-induced decreased saliva flow rate and body weight loss could be alleviated by nitrate supplementation. Nitrate prevented acinar and microvascular endothelial cell loss. Moreover, nitrate improved mitochondrial function and significantly decreased pyroptosis-related indexes. In vitro, nitrate supplementation reduced reactive oxygen species (ROS) generation by preserving mitochondrial homeostasis to inhibit NLPR3 inflammasome-mediated pyroptosis both in HUVECs and SMG cells. Nitrate showed potential as an oral protective agent to prevent IR-induced salivary gland damage; prospective insight into the underlying molecular mechanisms is presented.
Purpose:
This study aims to elaborate on the current knowledge concerning the mechanism, frequency, clinical manifestations, diagnostic procedures, prevention, and management of radioactive iodine (RAI)-induced sialadenitis in patients receiving treatment for differentiated thyroid cancer (DTC).
Methods:
A review of the literature was carried out through the " www.ncbi.nlm.nih.gov/pubmed " database focusing on the results of the past decade.
Results:
The high concentration of RAI in the salivary glands results in high beta radiation exposure of the striated duct cells and stem cells. This exposure leads to acute and/or chronic sialadenitis with obstructive symptoms and progressive loss of salivary gland function and xerostomia, with severe impact on patients' quality of life. No standard diagnostic method has been established. As far as prevention is concerned, many approaches have been proposed, such as sialogogues, local massage, vitamin E, and amifostine administration. Although there is no unanimity as to their effectiveness, the use of sialogogues is recommended. Treatment includes conservative drug therapy and sialendoscopy when necessary.
Conclusion:
RAI-induced sialadenitis has a major impact on patients' quality of life. Due to the good prognosis of DTC, the reduction of sialadenitis and its prognosis, prevention, and treatment constitute a priority for the overall treatment of these patients. Further studies that will establish a coherent treatment protocol for this condition are necessary.
Background
C1q/tumour necrosis factor-related protein 3 (CTRP3) plays important roles in metabolism and inflammatory responses in various cells and tissues. However, the expression and function of CTRP3 in salivary glands have not been explored.
Methods
The expression and distribution of CTRP3 were detected by western blot, polymerase chain reaction, immunohistochemical and immunofluorescence staining. The effects of CTRP3 on tumour necrosis factor (TNF)-α-induced apoptosis and barrier dysfunction were detected by flow cytometry, western blot, co-immunoprecipitation, and measurement of transepithelial resistance and paracellular tracer flux.
Results
CTRP3 was distributed in both acinar and ductal cells of human submandibular gland (SMG) and was primarily located in the ducts of rat and mouse SMGs. TNF-α increased the apoptotic rate, elevated expression of cleaved caspase 3 and cytochrome C, and reduced B cell lymphoma-2 (Bcl-2) levels in cultured human SMG tissue and SMG-C6 cells, and CTRP3 further enhanced TNF-α-induced apoptosis response. Additionally, CTRP3 aggravated TNF-α-increased paracellular permeability. Mechanistically, CTRP3 promoted TNF-α-enhanced TNF type I receptor (TNFR1) expression, inhibited the expression of cellular Fas-associated death domain (FADD)-like interleukin-1β converting enzyme inhibitory protein (c-FLIP), and increased the recruitment of FADD with receptor-interacting protein kinase 1 and caspase 8. Moreover, CTRP3 was significantly increased in the labial gland of Sjögren's syndrome patients and in the serum and SMG of nonobese diabetic mice.
Conclusions
These findings suggest that the salivary glands are a novel source of CTRP3 synthesis and secretion. CTRP3 might promote TNF-α-induced cell apoptosis through the TNFR1-mediated complex II pathway.
Radiation therapy for head and neck cancer causes damage to the surrounding salivary glands, resulting in salivary gland hypofunction and xerostomia. Current treatments do not provide lasting restoration of salivary gland function following radiation; therefore, a new mechanistic understanding of the radiation-induced damage response is necessary for identifying therapeutic targets. The purpose of the present study was to investigate the metabolic phenotype of radiation-induced damage in parotid salivary glands by integrating transcriptomic and metabolomic data. Integrated data were then analyzed to identify significant gene-metabolite interactions. Mice received a single 5 Gy dose of targeted head and neck radiation. Parotid tissue samples were collected 5 days following treatment for RNA sequencing and metabolomics analysis. Altered metabolites and transcripts significantly converged on a specific region in the metabolic reaction network. Both integrative pathway enrichment using rank-based statistics and network analysis highlighted significantly coordinated changes in glutathione metabolism, energy metabolism (TCA cycle and thermogenesis), peroxisomal lipid metabolism, and bile acid production with radiation. Integrated changes observed in energy metabolism suggest that radiation induces a mitochondrial dysfunction phenotype. These findings validated previous pathways involved in the radiation-damage response, such as altered energy metabolism, and identified robust signatures in salivary glands, such as reduced glutathione metabolism, that may be driving salivary gland dysfunction.
Salivary glands sustain collateral damage following radiotherapy (RT) to treat cancers of the head and neck, leading to complications, including mucositis, xerostomia and hyposalivation. Despite salivary gland-sparing techniques and modified dosing strategies, long-term hypofunction remains a significant problem. Current therapeutic interventions provide temporary symptom relief, but do not address irreversible glandular damage. In this review, we summarize the current understanding of mechanisms involved in RT-induced hyposalivation and provide a framework for future mechanistic studies. One glaring gap in published studies investigating RT-induced mechanisms of salivary gland dysfunction concerns the effect of irradiation on adjacent non-irradiated tissue via paracrine, autocrine and direct cell–cell interactions, coined the bystander effect in other models of RT-induced damage. We hypothesize that purinergic receptor signaling involving P2 nucleotide receptors may play a key role in mediating the bystander effect. We also discuss promising new therapeutic approaches to prevent salivary gland damage due to RT.
The tetraspan plasma membrane protein PERP (p53 apoptosis effector related to PMP22) is a lesser-known transcriptional target of p53 and p63. A member of the PMP22/GAS3/EMP membrane protein family, PERP was originally identified as a p53 target specifically trans-activated during apoptosis, but not during cell-cycle arrest. Several studies have since shown downregulation of PERP expression in numerous cancers, suggesting that PERP is a tumour suppressor protein. This review focusses on the important advances made in elucidating the mechanisms regulating PERP expression and its function as a tumour suppressor in diverse human cancers, including breast cancer and squamous cell carcinoma. Investigating PERP's role in clinically-aggressive uveal melanoma has revealed that PERP engages a positive-feedback loop with p53 to regulate its own expression, and that p63 is required beside p53 to achieve pro-apoptotic levels of PERP in this cancer. Furthermore, the recent discovery of apoptosis-mediating interaction of PERP with SERCA2b at the plasma membrane-endoplasmic reticulum interface demonstrates a novel mechanism of PERP stabilisation, and how PERP can mediate Ca²⁺ signalling to facilitate apoptosis. The multi-faceted role of PERP in cancer, involving well-documented functions in mediating apoptosis and cell-cell adhesion is discussed, alongside emerging roles in epithelial-mesenchymal transition, PERP's crosstalk with inflammation pathways, and other signalling pathways. The potential for restoring PERP expression as a means of cancer therapy is also considered.
IR irradiation‐induced dysfunction of the salivary gland and hyposalivation are one of the main complications of radiotherapy treatment for malignant head and neck tumors, and its mechanisms are not fully understood. IR‐induced microcirculation disturbance and apoptosis is one of the most important mechanisms. Most recent studies have focused on the regenerative effects of epoxyeicosatrienoic acids (EETs) through the process of increased angiogenesis. This study suggests that increasing endogenous EETs by the soluble epoxide hydrolase (sEH) inhibitor TPPU can reverse dysfunctional salivary glands and hyposalivation by improving the associated microenvironment and tissue regeneration. The findings suggest that TPPU alleviates hyposalivation and histological lesions, increases angiogenesis and reduces apoptosis of acinar cells significantly in IR‐damaged mice. The results in vitro demonstrate that TPPU promotes the proliferation and inhibits apoptosis of human salivary gland (HSG) cells, and enhances the motility and angiogenesis of human umbilical vein endothelial cells (HUVECs). The results also suggest that TPPU‐induced synergy and crosstalk between acinar cells and vascular endothelial cells might contribute to angiogenesis. Together, this study proves the feasibility of targeting sEH using TPPU to promote angiogenesis and reduce apoptosis, and also indicates the potential of sEH inhibitors for functional reconstruction of IR‐induced salivary glands damage.
Purpose:
To explore the effects and mechanisms of paracrine factors secreted from human adipose mesenchymal stem cells (hAdMSCs) that are activated by hypoxia on radioprotection against irradiation-induced salivary hypofunction in subjects undergoing radiotherapy for head and neck cancers.
Materials and methods:
An organotypic spheroid coculture model to mimic IR-induced salivary hypofunction was set up for in vitro experiments. Human parotid gland epithelial cells were organized to form three-dimensional acinus-like spheroids on growth factor reduced (GFR)-Matrigel. Cellular, structural, and functional damage following irradiation were examined after cells were cocultured with hAdMSCs preconditioned with either normoxia (hAdMSCNMX) or hypoxia (hAdMSCHPX). A key paracrine factor secreted by hAdMSCsHPXwas identified by high-throughput microarray-based enzyme-linked immunosorbent assay. Molecular mechanisms and signaling pathways on radioprotection were explored. Therapeutic effects of hAdMSCsHPXwere evaluated after in vivo transplant into mice with irradiation-induced salivary hypofunction.
Results:
In our 3D coculture experiment, hAdMSCsHPXsignificantly enhanced radioresistance of spheroidal hPECs, and led to greater preservation of salivary epithelial integrity and acinar secretory function relative to hAdMSCsNMX. Coculture with hAdMSCsHPXpromoted FGFR expression and suppressed FGFR diminished antiapoptotic activity of hAdMSCsHPX. Among FGFR-binding secreted factors, we found that FGF10 contributed to therapeutic effects of hAdMSCsHPXby enhancing antiapoptotic effect, which was dependent on FGFR-PI3K signaling. An in vivo transplant of hAdMSCsHPXinto irradiated salivary glands of mice reversed IR-induced salivary hypofunction where hAdMSC-released FGF10 contributed to tissue remodeling.
Conclusion:
Our results suggest that hAdMSCsHPXprotect salivary glands from irradiation-induced apoptosis, and preserve acinar structure and functions by activation of FGFR-PI3K signaling via actions of hAdMSC-secreted factors, including FGF10. This article is protected by copyright. All rights reserved.
Acute and chronic salivary gland dysfunction are common sequelae of radiotherapy for head and neck cancer; but the associated morphologic changes, especially of the acute damage, have received relatively little study. For investigation of the morphologic characteristics of acute radiation injury to parotid glands, rhesus monkeys were studied 1-72 hours after parotid irradiation with single doses of 2.5-15.0 Gy. The acute damage from all doses was clearly expressed by 24 hours. Histologically, parotid glands irradiated with 2.5 or 5.0 Gy had random degeneration and necrosis of the serous acinar cells. Doses of 7.5-15.0 Gy produced widespread degeneration along with necrosis of whole acini. Serous cell damage was accompanied by neutrophilic inflammation that subsided after 24 hours to become replaced by plasma cell and lymphocytic infiltrates. Parotid glands receiving 7.5-15.0 Gy were atrophic at 16-22 weeks after irradiation and showed no recovery by 40 weeks. Although parotid acinar cells are well-differentiated nondividing cells, these observations show that they express lethal radiation injury in interphase within hours of receiving a radiation dose as low as 2.5 Gy. This is unlike most mammalian cells that express radiation injury during mitosis. Chronic atrophy is a consequence of this direct, irreversible, and early injury, rather than the result of radiation-induced changes in the vasculature.
The tumor suppressor p53 is a cell cycle checkpoint protein that contributes to the preservation of genetic stability by mediating either a G1 arrest or apoptosis in response to DNA damage. Recent reports suggest that p53 causes growth arrest through transcriptional activation of the cyclin-dependent kinase (Cdk)-inhibitor Cip1. Here, we characterize the p53-dependent G1 arrest in several normal human diploid fibroblast (NDF) strains and p53-deficient cell lines treated with 0.1-6 Gy gamma radiation. DNA damage and cell cycle progression analyses showed that NDF entered a prolonged arrest state resembling senescence, even at low doses of radiation. This contrasts with the view that p53 ensures genetic stability by inducing a transient arrest to enable repair of DNA damage, as reported for some myeloid leukemia lines. Gamma radiation administered in early to mid-, but not late, G1 induced the arrest, suggesting that the p53 checkpoint is only active in G1 until cells commit to enter S phase at the G1 restriction point. A log-linear plot of the fraction of irradiated G0 cells able to enter S phase as a function of dose is consistent with single-hit kinetics. Cytogenetic analyses combined with radiation dosage data indicate that only one or a small number of unrepaired DNA breaks may be sufficient to cause arrest. The arrest also correlated with long-term elevations of p53 protein, Cip1 mRNA, and Cip1 protein. We propose that p53 helps maintain genetic stability in NDF by mediating a permanent cell cycle arrest through long-term induction of Cip1 when low amounts of unrepaired DNA damage are present in G1 before the restriction point.
To analyze the involvement of p53-dependent transcriptional activation in normal development and in response to DNA damage in vivo, we created transgenic mice with a lacZ reporter gene under the control of a p53-responsive promoter. Five independent strains showed similar patterns of transgene expression. In untreated animals, lacZ expression was limited to the developing nervous system of embryos and newborn mice and was strongly decreased in the adult brain. gamma-irradiation or adriamycin treatment induced lacZ expression in the majority of cells of early embryos and in the spleen, thymus and small intestine in adult mice. Transgene expression was p53 dependent and coincided with the sites of strong p53 accumulation. The lacZ-expressing tissues and early embryos, unlike other adult tissues and late embryos, are characterized by high levels of p53 mRNA expression and respond to DNA damage by massive apoptotic cell death. Analysis of p53-null mice showed that this apoptosis is p53 dependent. These data suggest that p53 activity, monitored by the reporter lacZ transgene, is the determinant of radiation and drug sensitivity in vivo and indicate the importance of tissue and stage specificity of p53 regulation at the level of mRNA expression.
Chemotherapy and radiation therapy for cancer often have severe side effects that limit their efficacy. Because these effects
are in part determined by p53-mediated apoptosis, temporary suppression of p53 has been suggested as a therapeutic strategy
to prevent damage of normal tissues during treatment of p53-deficient tumors. To test this possibility, a small molecule was
isolated for its ability to reversibly block p53-dependent transcriptional activation and apoptosis. This compound, pifithrin-α,
protected mice from the lethal genotoxic stress associated with anticancer treatment without promoting the formation of tumors.
Thus, inhibitors of p53 may be useful drugs for reducing the side effects of cancer therapy and other types of stress associated
with p53 induction.
Autophagy, the process by which cells recycle cytoplasm and dispose of excess or defective organelles, has entered the research
spotlight largely owing to the discovery of the protein components that drive this process. Identifying the autophagy genes
in yeast and finding orthologs in other organisms reveals the conservation of the mechanism of autophagy in eukaryotes and
allows the use of molecular genetics and biology in different model systems to study this process. By mostly morphological
studies, autophagy has been linked to disease processes. Whether autophagy protects from or causes disease is unclear. Here,
we summarize current knowledge about the role of autophagy in disease and health.
Cell growth and proliferation requires an intricate coordination between the stimulatory signals arising from nutrients and growth factors and the inhibitory signals arising from intracellular and extracellular stresses. Alteration of the coordination often causes cancer. In mammals, the mTOR (mammalian target of rapamycin) protein kinase is the central node in nutrient and growth factor signaling, and p53 plays a critical role in sensing genotoxic and other stresses. The results presented here demonstrate that activation of p53 inhibits mTOR activity and regulates its downstream targets, including autophagy, a tumor suppression process. Moreover, the mechanisms by which p53 regulates mTOR involves AMP kinase activation and requires the tuberous sclerosis (TSC) 1/TSC2 complex, both of which respond to energy deprivation in cells. In addition, glucose starvation not only signals to shut down mTOR, but also results in the transient phosphorylation of the p53 protein. Thus, p53 and mTOR signaling machineries can cross-talk and coordinately regulate cell growth, proliferation, and death.
• AMP-activated kinase
• autophagy
• tuberous sclerosis 1/tuberous sclerosis 2
Protein kinase C (PKC) δ is an essential regulator of mitochondrial dependent apoptosis in epithelial cells. We have used
the PKCδ-/- mouse to ask if loss of PKCδ protects salivary glands against γ-irradiation-induced apoptosis in vivo and to explore the mechanism underlying protection from apoptosis. We show that γ-irradiation in vivo results in a robust induction of apoptosis in the parotid glands of wild type mice, whereas apoptosis is suppressed by greater
than 60% in the parotid glands of PKCδ-/- mice. Primary parotid cells from PKCδ-/- mice are defective in mitochondrial dependent apoptosis as indicated by suppression of etoposide-induced cytochrome c release, poly(ADP-ribose) polymerase cleavage, and caspase-3 activation. Notably, apoptotic responsiveness can be restored
by re-introduction of PKCδ by adenoviral transduction. Etoposide and γ-irradiation-induced activation of p53 is similar in
primary parotid cells and parotid glands from PKCδ+/+ and PKCδ-/- mice, indicating that PKCδ functions downstream of the DNA damage response. In contrast, activation of the c-Jun amino-terminal
kinase is reduced in primary parotid cells from PKCδ-/- cells and in parotid C5 cells, which express a dominant inhibitory mutant of PKCδ. Similarly, c-Jun amino-terminal kinase
activation is suppressed in vivo in γ-irradiated parotid glands from PKCδ-/- mice. These studies indicate an essential role for PKCδ downstream of the p53 response and upstream of the c-Jun amino-terminal
kinase activation in DNA damage-induced apoptosis in vivo and in vitro.
Chronic damage to the salivary glands is a common side effect following head and neck irradiation. It is hypothesized that irreversible damage to the salivary glands occurs immediately after radiation; however, previous studies with rat models have not shown a causal role for apoptosis in radiation-induced injury. We report that etoposide and gamma irradiation induce apoptosis of salivary acinar cells from FVB control mice in vitro and in vivo; however, apoptosis is reduced in transgenic mice expressing a constitutively activated mutant of Akt1 (myr-Akt1). Expression of myr-Akt1 in the salivary glands results in a significant reduction in phosphorylation of p53 at serine(18), total p53 protein accumulation, and p21(WAF1) or Bax mRNA following etoposide or gamma irradiation of primary salivary acinar cells. The reduced level of p53 protein in myr-Akt1 salivary glands corresponds with an increase in MDM2 phosphorylation in vivo, suggesting that the Akt/MDM2/p53 pathway is responsible for suppression of apoptosis. Dominant-negative Akt blocked phosphorylation of MDM2 in salivary acinar cells from myr-Akt1 transgenic mice. Reduction of MDM2 levels in myr-Akt1 primary salivary acinar cells with small interfering RNA increases the levels of p53 protein and renders these cells susceptible to etoposide-induced apoptosis in spite of the presence of activated Akt1. These results indicate that MDM2 is a critical substrate of activated Akt1 in the suppression of p53-dependent apoptosis in vivo.
p73, an important developmental gene, shares a high sequence homology with p53 and induces both G(1) cell cycle arrest and apoptosis. However, the molecular mechanisms through which p73 induces apoptosis are unclear. We found that p73-induced apoptosis is mediated by PUMA (p53 up-regulated modulator of apoptosis) induction, which, in turn, causes Bax mitochondrial translocation and cytochrome c release. Overexpression of p73 isoforms promotes cell death and bax promoter transactivation in a time-dependent manner. However, the kinetics of apoptosis do not correlate with the increase of Bax protein levels. Instead, p73-induced mitochondrial translocation of Bax is kinetically compatible with the induction of cell death. p73 is localized in the nucleus and remains nuclear during the induction of cell death, indicating that the effect of p73 on Bax translocation is indirect. The ability of p73 to directly transactivate PUMA and the direct effect of PUMA on Bax conformation and mitochondrial relocalization suggest a molecular link between p73 and the mitochondrial apoptotic pathway. Our data therefore indicate that PUMA-mediated Bax mitochondrial translocation, rather than its direct transactivation, correlates with cell death. Finally, human DeltaNp73, an isoform lacking the amino-terminal transactivation domain, inhibits TAp73-induced as well as p53-induced apoptosis. The DeltaNp73 isoforms seem therefore to act as dominant negatives, repressing the PUMA/Bax system and, thus, finely tuning p73-induced apoptosis. Our findings demonstrate that p73 elicits apoptosis via the mitochondrial pathway using PUMA and Bax as mediators.
Learning Objectives
After completing this course, the reader will be able to: Outline innovations related to targeted radiation therapy.Describe trials proving an advantage using IMRT.Assess treatment planning modalities and how IMRT fields are designed.Evaluate when there is a toxicity advantage for IMRT.Discern when conventional radiotherapy should be used instead of IMRT.
CME Access and take the CME test online and receive 1 AMA PRA Category 1 Credit™ at CME.TheOncologist.com
The aim of this study was to perform a literature review about the composition and functions of saliva as well as describe the factors that influence salivary flow (SF) and its biochemical composition.
Saliva represents an increasingly useful auxiliary means of diagnosis. Sialometry and sialochemistry are used to diagnose systemic illnesses, monitoring general health, and as an indicator of risk for diseases creating a close relation between oral and systemic health.
This review provides fundamental information about the salivary system in terms of normal values for SF and composition and a comprehensive review of the factors that affect this important system.
Since several factors can influence salivary secretion and composition, a strictly standardized collection must be made so the above-mentioned exams are able to reflect the real functioning of the salivary glands and serve as efficient means for monitoring health.
Since many oral and systemic conditions manifest themselves as changes in the flow and composition of saliva the dental practitioner is advised to remain up-to-date with the current literature on the subject.
The p53 protein regulates the transcription of many different genes in response to a wide variety of stress signals. Following DNA damage, p53 regulates key processes, including DNA repair, cell-cycle arrest, senescence and apoptosis, in order to suppress cancer. This Analysis article provides an overview of the current knowledge of p53-regulated genes in these pathways and others, and the mechanisms of their regulation. In addition, we present the most comprehensive list so far of human p53-regulated genes and their experimentally validated, functional binding sites that confer p53 regulation.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-DeltaDeltaCr) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-DeltaDeltaCr) method. In addition, we present the derivation and applications of two variations of the 2(-DeltaDeltaCr) method that may be useful in the analysis of real-time, quantitative PCR data. (C) 2001 Elsevier science.
Through direct cloning of p53 binding sequences from human genomic DNA, we have isolated a novel gene, designated p53AIP1 (p53-regulated Apoptosis-Inducing Protein 1), whose expression is inducible by wild-type p53. Ectopically expressed p53AIP1, which is localized within mitochondria, leads to apoptotic cell death through dissipation of mitochondrial ΔΨm. We have found that upon severe DNA damage, Ser-46 on p53 is phosphorylated and apoptosis is induced. In addition, substitution of Ser-46 inhibits the ability of p53 to induce apoptosis and selectively blocks expression of p53AIP1. Our results suggest that p53AIP1 is likely to play an important role in mediating p53-dependent apoptosis, and phosphorylation of Ser-46 regulates the transcriptional activation of this apoptosis-inducing gene.
The aim of this study was to monitor composition and rate of secretion of rat parotid and submandibular/sublingual saliva following local single doses of X-rays ranging from 5 to 20 Gy. Pilocarpine-stimulated samples of parotid and submandibular/sublingual saliva were simultaneously collected with miniaturized Lashley cups before and 1-30 days after irradiation. The lag phase (period between injection of pilocarpine and start of the secretion) and flow rate were recorded and the concentrations of sodium, potassium, calcium, phosphate, and amylase were measured. With increasing dose and time, the salivary flow rate as well as sodium concentration decreased, while potassium concentrations increased throughout the follow-up period. The lag phase and the concentration of amylase reached their maximum at 3 and 10 days after irradiation, respectively. The changes in lag phase and flow rate were most obvious after doses of 15 or 20 Gy and showed a great similarity for parotid and submandibular/sublingual saliva. No dose-response relationship was observed for the changes in concentrations of calcium and phosphate. It is concluded that for radiation doses of 10 Gy and above, irreversible changes (lag phase, flow rate, potassium, sodium) were observed. A saturation of the irradiation effects (lag phase, flow rate) seems to exist at doses larger than 15 Gy. No significant differences were observed between the radiation-induced functional changes in parotid and submandibular/sublingual salivary gland tissue.
Saliva is important for the preservation and maintenance of oral health. It is unclear, however, how much saliva is required to maintain normal oral function. Major salivary gland flow rates, objective measurements of oral health, and subjective complaints of oral problems were assessed in different-aged, healthy persons. Results suggest that the comparison of major salivary gland flow rates of an individual with population standards to identify patients susceptible to the effects of salivary dysfunction is unreliable. Changes in salivary function over time are a more meaningful gauge of the impact of saliva on oral health. The clinician should monitor salivary production to identify patients with declining salivary gland output.
To find an answer to the question: Are the acute radiation effects on salivary gland function, as seen in earlier studies, causally related to radiation-induced apoptosis?
Rat parotid and submandibular glands were X-irradiated with doses up to 25 Gy and morphological damage assayed up to 6 days after irradiation. Damage to the different cell types in the glands was assessed after H & E staining. Apoptotic appearance was judged by compacted chromatin and fragmentation of cells into lobulated masses.
In about 3% of the cells aberrant nuclei were observed after doses as low as 2 Gy and around 7.5 and 24 h after irradiation. About half of these aberrant nuclei had an apoptotic appearance. After a dose of about 5 Gy no dose-response for apoptotic cells was found, as evidenced by a plateau in the dose-effect curve. At 6 days after 2 Gy, no signs of radiation-induced apoptosis was apparent and for most cell types a value close to zero was observed.
Radiation studies on salivary function in the rat show the typical response with respect to dose (5-15 Gy) and time (1-3 days). This differs from reported findings with light microscopy. Therefore, the extent of apoptosis induced by radiation cannot explain the observed gland malfunction. Alternative mechanisms are proposed.
The effect of a single dose of X-irradiation (either 2.5, 5, 7.5, 10 or 15 Gy) to the head and neck region on parotid and submandibular gland function in rats was evaluated for up to 1 year. No animal receiving 15 Gy survived the entire study. Animals receiving > or = 7.5 Gy showed significantly less increase in body weight over time. Average wet weights of both gland types were reduced with as little as 2.5 Gy. Pilocarpine-stimulated parotid salivary flow was diminished significantly at 12 months for each radiation-dose group. Significant salivary flow reductions from submandibular glands were seen at > or = 7.5 Gy at this same time-point. These results show that a single radiation exposure of as low as 2.5 Gy to the head and neck region of rats can cause significant long-term alterations in salivary gland function.
To determine the relationships between the three-dimensional dose distributions in parotid glands and their saliva production, and to find the doses and irradiated volumes that permit preservation of the salivary flow following irradiation (RT).
Eighty-eight patients with head and neck cancer irradiated with parotid-sparing conformal and multisegmental intensity modulation techniques between March 1994 and August 1997 participated in the study. The mean dose and the partial volumes receiving specified doses were determined for each gland from dose-volume histograms (DVHs). Nonstimulated and stimulated saliva flow rates were selectively measured from each parotid gland before RT and at 1, 3, 6, and 12 months after the completion of RT. The data were fit using a generalized linear model and the normal tissue complication probability (NTCP) model of Lyman-Kutcher. In the latter model, a "severe complication" was defined as salivary flow rate reduced to < or =25% pre-RT flow at 12 months.
Saliva flow rates data were available for 152 parotid glands. Glands receiving a mean dose below or equal to a threshold (24 Gy for the unstimulated and 26 Gy for the stimulated saliva) showed substantial preservation of the flow rates following RT and continued to improve over time (to median 76% and 114% of pre-RT for the unstimulated and stimulated flow rates, respectively, at 12 months). In contrast, most glands receiving a mean dose higher than the threshold produced little saliva with no recovery over time. The output was not found to decrease as mean dose increased, as long as the threshold dose was not reached. Similarly, partial volume thresholds were found: 67%, 45%, and 24% gland volumes receiving more than 15 Gy, 30 Gy, and 45 Gy, respectively. The partial volume thresholds correlated highly with the mean dose and did not add significantly to a model predicting the saliva flow rate from the mean dose and the time since RT. The NTCP model parameters were found to be TD50 (the tolerance dose for 50% complications rate for whole organ irradiated uniformly) = 28.4 Gy, n (volume dependence parameter) = 1, and m (the slope of the dose/response relationship) = 0.18. Clinical factors including age, gender, pre-RT surgery, chemotherapy, and certain medical conditions were not found to be significantly associated with the salivary flow rates. Medications (diuretics, antidepressants, and narcotics) were found to adversely affect the unstimulated but not the stimulated flow rates.
Dose/volume/function relationships in the parotid glands are characterized by dose and volume thresholds, steep dose/response relationships when the thresholds are reached, and a maximal volume dependence parameter in the NTCP model. A parotid gland mean dose of < or =26 Gy should be a planning goal if substantial sparing of the gland function is desired.
Breast cancer is the most frequent tumor type among women in the United States and in individuals with Li-Fraumeni syndrome. The p53 tumor suppressor gene is altered in a large proportion of both spontaneous breast malignancies and Li-Fraumeni breast cancers. This suggests that loss of p53 can accelerate breast tumorigenesis, yet p53-deficient mice rarely develop mammary tumors. To evaluate the effect of p53 loss on mammary tumor formation, the p53(null) allele was back-crossed onto the BALB/c genetic background. Median survival was 15.4 weeks for BALB/c-p53(-/-) mice compared to 54 weeks for BALB/c-p53(+/-) mice. Sarcomas and lymphomas were the most frequent tumor types in BALB/c-p53(-/-) mice, whereas 55% of the female BALB/c-p53(+/-) mice developed mammary carcinomas. The mammary tumors were highly aneuploid, frequently lost the remaining wild-type p53 allele, but rarely lost BRCA1. Although mammary tumors were rarely detected in BALB/c-p53(-/-) female mice, when glands from BALB/c-p53(-/-) mice were transplanted into wild-type BALB/c hosts, 75% developed mammary tumors. The high rate of mammary tumor development in the BALB/c background, but not C57Bl/6 or 129/Sv, suggests a genetic predisposition toward mammary tumorigenesis. Therefore, the BALB/c-p53(+/-) mice provide a unique model for the study of breast cancer in Li-Fraumeni syndrome. These results demonstrate the critical role that the p53 tumor suppressor gene plays in preventing tumorigenesis in the mammary gland.
Activation of the antiapoptotic protein kinase Akt is induced by a number of growth factors that regulate mammary gland development. Akt is expressed during mammary gland development, and expression decreases at the onset of involution. To address Akt actions in mammary gland development, transgenic mice were generated expressing constitutively active Akt in the mammary gland under the control of the mouse mammary tumor virus (MMTV) promoter. Analysis of mammary glands from these mice reveals a delay in both involution and the onset of apoptosis. Expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), an inhibitor of matrix metalloproteinases (MMPs), is prolonged and increased in the transgenic mice, suggesting that disruption of the MMP:TIMP ratio may contribute to the delayed mammary gland involution observed in the transgenic mice.
To assess long-term xerostomia in patients receiving parotid-sparing radiation therapy (RT) for head-and-neck cancer, and to find the patient and therapy-related factors that affect its severity.
From March 1994 through January 2000, 84 patients received comprehensive bilateral neck RT using conformal and multisegmental intensity-modulated RT (IMRT) aiming to spare the major salivary glands. Before RT and periodically through 2 years after the completion of RT, salivary flow rates from each of the major salivary glands were selectively measured. At the same time intervals, each patient completed an 8-item self-reported xerostomia-specific questionnaire (XQ). To gain a relative measure of the effect of RT on the minor salivary glands, whose output could not be measured, the surfaces of the oral cavity (extending to include the surface of the base of tongue) were outlined in the planning CT scans. The mean doses to the new organ ("oral cavity") were recorded. Forty-eight patients receiving unilateral neck RT were similarly studied and served as a benchmark for comparison. Factors predicting the XQ scores were analyzed using a random-effects model.
The XQ was found to be reliable and valid in measuring patient-reported xerostomia. The spared salivary glands which had received moderate doses in the bilateral RT group recovered to their baseline salivary flow rates during the second year after RT, and the spared glands in the unilateral RT group, which had received very low doses, demonstrated increased salivary production beyond their pre-RT levels. The increase in the salivary flow rates during the second year after RT paralleled an improvement in xerostomia in both patient groups. The improvement in xerostomia was faster in the unilateral compared with the bilateral RT group, but the difference narrowed at 2 years. The major salivary gland flow rates had only a weak correlation with the xerostomia scores. Factors found to be independently associated with the xerostomia scores were the pre-RT baseline scores, the time since RT, and the mean doses to the major salivary glands (notably to the submandibular glands) and to the oral cavity.
An improvement over time in xerostomia, occurring in tandem with rising salivary production from the spared major salivary glands, suggests a long-term clinical benefit from their sparing. The oral cavity mean dose, representing RT effect on the minor salivary glands, was found to be a significant, independent predictor of xerostomia. Thus, in addition to the major salivary glands, sparing the uninvolved oral cavity should be considered as a planning objective to further reduce xerostomia.
HER-2/neu amplification or overexpression can make cancer cells resistant to apoptosis and promotes their growth. p53 is crucial in regulating cell growth and apoptosis, and is often mutated or deleted in many types of tumour. Moreover, many tumours with a wild-type gene for p53 do not have normal p53 function, suggesting that some oncogenic signals suppress the function of p53. In this study, we show that HER-2/neu-mediated resistance to DNA-damaging agents requires the activation of Akt, which enhances MDM2-mediated ubiquitination and degradation of p53. Akt physically associates with MDM2 and phosphorylates it at Ser166 and Ser186. Phosphorylation of MDM2 enhances its nuclear localization and its interaction with p300, and inhibits its interaction with p19ARF, thus increasing p53 degradation. Our study indicates that blocking the Akt pathway mediated by HER-2/neu would increase the cytotoxic effect of DNA-damaging drugs in tumour cells with wild-type p53.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.
Ionizing radiation (IR) has proven to be a powerful medical treatment in the fight against cancer. Rational and effective use of its killing power depends on understanding IR-mediated responses at the molecular, cellular and tissue levels. Tumour cells frequently acquire defects in the molecular regulatory mechanisms of the response to IR, which sensitizes them to radiation therapy. One of the key molecules involved in a cell's response to IR is p53. Understanding these mechanisms indicates new rational approaches to improving cancer treatment by IR.
We have explored the interactions between the NFkappaB and Cdk-Rb-E2F pathways in controlling T cell fate following antigen stimulation. The inhibition of NFkappaB in antigen-stimulated T cells results in apoptosis but does not inhibit E2F activation and S phase entry. IkappaB-induced apoptosis coincides with the superinduction of p73 expression and activity. G1 Cdk activity is required for IkappaB-induced apoptosis and the induction of p73. Importantly, p73 deficiency rescues activated T cells from the apoptosis resulting from the inhibition of NFkappaB. Thus, Cdk2 activation sends signals for both cell cycle progression and apoptosis, the latter of which must be blocked by NFkappaB to allow for proliferation.
p53 is a master regulator of cell death pathways and has transcription-dependent and transcription- independent modes of action. Mitochondria are major signal transducers in apoptosis and are critical for p53-dependent cell death. Recently, we discovered that a fraction of stress-induced wild-type p53 protein rapidly translocates to mitochondria during p53-dependent apoptosis. Suborganellar localization by various methods shows that p53 predominantly localizes to the surface of mitochondria. Moreover, bypassing the nucleus by targeting p53 to mitochondria is sufficient to induce apoptosis in p53-null cells, without requiring further DNA damage. Here, we describe subcellular fractionation as a classic technique for detecting mitochondrial p53 in cell extracts. It consists of cell homogenization by hypo-osmotic swelling, removal of nuclear components by low-speed centrifugation, and mitochondrial isolation by a discontinuous sucrose density gradient. p53 and other mitochondrial proteins can then be detected by standard immunoblotting procedures. The quality of mitochondrial isolates can be verified for purity and intactness.
The induction of apoptosis by the p53 protein is critical for its activity as a tumor suppressor. Although it is clear that p53 induces apoptosis at least in part by direct transcriptional activation of target genes, the set of p53 target genes that mediate p53 function in apoptosis in vivo remains to be well defined. The Perp (p53 apoptosis effector related to PMP-22) gene is highly expressed in cells undergoing p53-dependent apoptosis as compared to cells undergoing p53-dependent G1 arrest. Perp is a direct p53 target, and its overexpression is sufficient to induce cell death in fibroblasts, implicating it as an important component of p53 apoptotic function. Here, through the generation of Perp-deficient mice, we analyze the role of Perp in the p53 apoptosis pathway in multiple primary cell types by comparing the cell death responses of Perp null cells to those of wild-type and p53 null cells. These experiments demonstrate the involvement of Perp in p53-mediated cell death in thymocytes and neurons but not in E1A-expressing MEFs, indicating a cell type-specific role for Perp in the p53 cell death pathway. In addition, we show that Perp is not required for proliferation-associated functions of p53. Thus, Perp selectively mediates the p53 apoptotic response, and the requirement for Perp is dictated by cellular context.
The mechanism by which some BH3-only proteins of the Bcl-2 family directly activate the "multidomain" proapoptotic member Bax is poorly characterized. We report that the first alpha helix (Halpha1) of Bax specifically interacts with the BH3 domains of Bid and PUMA but not with that of Bad. Inhibition of this interaction, by a peptide comprising Halpha1 or by a mutation in this helix, prevents ligand-induced activation of Bax by Bid, PUMA, or their BH3 peptides. Halpha1-mutated Bax, which can mediate death induced by Bad or its BH3 peptide, does not mediate that induced by Bid, PUMA, or their BH3 peptides. The response of Halpha1-mutated Bax to Bid can be restored by a compensating mutation in Bid BH3. Thus, a specific interaction between Bax Halpha1 and their BH3 domains allows Bid and PUMA to function as "death agonists" of Bax, whereas Bad recruits Bax activity through a distinct pathway.
Induction of apoptosis is one of the central activities by which p53 exerts its tumor-suppressing function. Aside from its primary function as a transcription factor, it can promote apoptosis independent of transcription. Recent studies have started to define the mechanisms of non-transcriptional pro-apoptotic p53 activities operating within the intrinsic mitochondria-mediated pathway of apoptosis. So far, two different mechanisms have been described, each of which was assigned to a specific localization of the p53 protein, either in the cytosol or directly at the mitochondria. Although mechanistically different, both transcription-independent modes of apoptosis induction converge, as they both initiate permeabilization of the outer mitochondrial membrane via activation of the pro-apoptotic Bcl-2 family members Bax or Bak.
Complex proapoptotic functions are essential for the tumor suppressor activity of p53. We recently described a novel transcription-independent mechanism that involves a rapid proapoptotic action of p53 at the mitochondria and executes the shortest known circuitry of p53 death signaling. Here, we examine if this p53-dependent mitochondrial program could be exploited for tumor suppression in vivo. To test this, we engage Emu-Myc transgenic mice, a well-established model of p53-dependent lymphomagenesis. We show that exclusive delivery of p53 to the outer mitochondrial membrane confers a significant growth disadvantage on Emu-Myc-transformed B-cells of p53-deficient or alternate reading frame-deficient genotypes, resulting in efficient induction of apoptosis and impinged proliferation. Conversely, normal cells from thymus, spleen, and bone marrow showed poor infectivity with these viruses. This proof-of-principle experiment shows that exclusive reliance on the direct mitochondrial program exerts a significant tumor suppressor activity in vivo. Our in vivo data on the direct mitochondrial apoptotic p53 program lays the groundwork to further investigate its efficacy and safety and to address its possible therapeutic value in the future.
Defective apoptosis renders immortalized epithelial cells highly tumorigenic, but how this is impacted by other common tumor mutations is not known. In apoptosis-defective cells, inhibition of autophagy by AKT activation or by allelic disruption of beclin1 confers sensitivity to metabolic stress by inhibiting an autophagy-dependent survival pathway. While autophagy acts to buffer metabolic stress, the combined impairment of apoptosis and autophagy promotes necrotic cell death in vitro and in vivo. Thus, inhibiting autophagy under conditions of nutrient limitation can restore cell death to apoptosis-refractory tumors, but this necrosis is associated with inflammation and accelerated tumor growth. Thus, autophagy may function in tumor suppression by mitigating metabolic stress and, in concert with apoptosis, by preventing death by necrosis.
Autophagy was recently established as a novel tumor suppression mechanism, which stimulated a wave of investigations that were aimed at understanding exactly how autophagy prevents tumorigenesis, as well as to determine to what extent autophagy is implicated in human cancers. Autophagy might exert its tumor suppression function at the subcellular level by removing defective cytoplasmic components, such as damaged mitochondria. In addition, it might function at the cellular level by helping in the orderly removal of damaged cells. Previous studies indicated that autophagy is compromised in human breast, ovarian and prostate cancers. Recent research revealed that autophagy is activated by p53, a critical tumor suppressor that is involved in most, if not all, tumorigenesis. This study places autophagy in a broader context of human cancers. Future work elucidating the role of autophagy in the p53 circuit and p53 function might provide more insight into tumorigenesis and targeted cancer chemotherapy.
A common side effect experienced by head and neck cancer patients after radiation therapy (RT) is impairment of the parotid glands' ability to produce saliva. Our purpose is to investigate the relationship between radiation dose and saliva changes in the 2 years after treatment.
The study population includes 142 patients treated with conformal or intensity-modulated radiotherapy. Saliva flow rates from 266 parotid glands are measured before and 1, 3, 6, 12, 18, and 24 months after treatment. Measurements are collected separately from each gland under both stimulated and unstimulated conditions. Bayesian nonlinear hierarchical models were developed and fit to the data.
Parotids receiving higher radiation produce less saliva. The largest reduction is at 1-3 months after RT followed by gradual recovery. When mean doses are lower (e.g., <25 Gy), the model-predicted average stimulated saliva recovers to pretreatment levels at 12 months and exceeds it at 18 and 24 months. For higher doses (e.g., >30 Gy), the stimulated saliva does not return to original levels after 2 years. Without stimulation, at 24 months, the predicted saliva is 86% of pretreatment levels for 25 Gy and <31% for >40 Gy. We do not find evidence to support that the overproduction of stimulated saliva at 18 and 24 months after low dose in 1 parotid gland is the result of low saliva production from the other parotid gland.
Saliva production is affected significantly by radiation, but with doses <25-30 Gy, recovery is substantial and returns to pretreatment levels 2 years after RT.
The serine/threonine kinase Akt, also known as protein kinase B (PKB), is a central node in cell signaling downstream of growth factors, cytokines, and other cellular stimuli. Aberrant loss or gain of Akt activation underlies the pathophysiological properties of a variety of complex diseases, including type-2 diabetes and cancer. Here, we review the molecular properties of Akt and the approaches used to characterize its true cellular targets. In addition, we discuss those Akt substrates that are most likely to contribute to the diverse cellular roles of Akt, which include cell survival, growth, proliferation, angiogenesis, metabolism, and migration.
The purpose of this review is to suggest an updated description of early and late irradiation effects on salivary glands in rodents. Based on this description, the mechanism of the underlying xerostomia will be discussed.
To examine the assumption that the so-called "irradiation" effects on the parotid gland of the rat during the first two weeks are actually mucositic effects and, thus, are transient, both function and partitution-coefficient parameters of the salivary glands were examined in both irradiated and pair-fed but not irradiated rats.
Various studies were performed in which irradiated and non-irradiated rats were examined at varying intervals up to one year post-irradiation. Head and neck irradiation resulted not only in dysfunction and tissue loss of the salivary glands but also in a systemic effect expressed as profound body weight loss.
Based on the literature available and on our own studies, we believe that one can suggest an overall mechanism for the damage induced by irradiation to the salivary glands. Our results have shown a mutual delayed expression of irradiation-induced damage in both parotid and submandibular, more evident in the parotid gland.
We have suggested the following mechanism for the parotid irradiation-induced specific damage: The injurious agents resulting in delayed serous cell death leading to the specific parotid radiosensitivity are transition, highly redox-active metal ions, such as Fe and Cu, associated with secretion granules.
To analyse clinical and dosimetric characteristics with regard to clinical constraints in head and neck cancer patients treated with intensity-modulated radiotherapy (IMRT).
Between August 2001 and July 2005, 75 patients with non-metastatic head and neck cancers were treated with IMRT with curative intent. Dose-volume histograms (DVH) drawn up from inverse dosimetry were analysed and compared to the prescription according to the clinical presentation of the disease.
For bilateral irradiation, the mean doses delivered to the contralateral and ipsilateral parotid glands were, respectively, 28.1 and 26.3Gy. Dose constraints to the submandibular glands were only respected for the contralateral gland during unilateral irradiation. For tumors located in paranasal sinuses, the maximal dose to the contralateral and ipsilateral optic nerves remained significantly lower than the constraint doses while the constraints for the anterior part of the eyes could not be respected.
Significant differences were observed concerning respect of the constraints applied to the parotid or to the submandibular glands for medial tumors. The respect of constraints for the organs at risk critically depends on the location and size of the primary tumor and on the definition of the CTV. The clinical impact has to be further evaluated.
Submandibular salivary glands (SMGs) dysfunction contributes to xerostomia after radiotherapy (RT) of head-and-neck (HN) cancer. We assessed SMG dose-response relationships and their implications for sparing these glands by intensity-modulated radiotherapy (IMRT).
A total of 148 HN cancer patients underwent unstimulated and stimulated SMG salivary flow rate measurements selectively from Wharton's duct orifices, before RT and periodically through 24 months after RT. Correlations of flow rates and mean SMG doses were modeled throughout all time points. IMRT replanning in 8 patients whose contralateral level I was not a target incorporated the results in a new cost function aiming to spare contralateral SMGs.
Stimulated SMG flow rates decreased exponentially by (1.2%)(Gy) as mean doses increased up to 39 Gy threshold, and then plateaued near zero. At mean doses < or =39 Gy, but not higher, flow rates recovered over time at 2.2%/month. Similarly, the unstimulated salivary flow rates decreased exponentially by (3%)(Gy) as mean dose increased and recovered over time if mean dose was <39 Gy. IMRT replanning reduced mean contralateral SMG dose by average 12 Gy, achieving < or =39 Gy in 5 of 8 patients, without target underdosing, increasing the mean doses to the parotid glands and swallowing structures by average 2-3 Gy.
SMG salivary flow rates depended on mean dose with recovery over time up to a threshold of 39 Gy. Substantial SMG dose reduction to below this threshold and without target underdosing is feasible in some patients, at the expense of modestly higher doses to some other organs.
Multiple cellular stressors, including activation of the tumour suppressor p53, can stimulate autophagy. Here we show that deletion, depletion or inhibition of p53 can induce autophagy in human, mouse and nematode cells subjected to knockout, knockdown or pharmacological inhibition of p53. Enhanced autophagy improved the survival of p53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels. Inhibition of p53 led to autophagy in enucleated cells, and cytoplasmic, not nuclear, p53 was able to repress the enhanced autophagy of p53(-/-) cells. Many different inducers of autophagy (for example, starvation, rapamycin and toxins affecting the endoplasmic reticulum) stimulated proteasome-mediated degradation of p53 through a pathway relying on the E3 ubiquitin ligase HDM2. Inhibition of p53 degradation prevented the activation of autophagy in several cell lines, in response to several distinct stimuli. These results provide evidence of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53.
Transgenic mice with p53-responsive lacZ: p53 activity varies dramatically dur-ing normal development and determines radiation and drug sen-sitivity in vivo
- Komarova Ea
- Mv Chernov
- R Franks
Komarova EA, Chernov MV, Franks R, et al. Transgenic mice with p53-responsive lacZ: p53 activity varies dramatically dur-ing normal development and determines radiation and drug sen-sitivity in vivo. EMBO J 1997;16:1391–1400.
p53-dependent apoptosis in salivary glands d
- J L Avila
p53-dependent apoptosis in salivary glands d J. L. AVILA et al.
Suppression of apoptosis in the PKCdelta null mouse in vivo
- Humphries Mj Kh Limesand
- Jc Schneider
Humphries MJ, Limesand KH, Schneider JC, et al. Suppression of apoptosis in the PKCdelta null mouse in vivo. J Biol Chem 2006;281:9728–9737.
The role of p53 in determining sensitivity to radiotherapy
- Gudkov
Gudkov AV, Komarova EA. The role of p53 in determining sensitivity to radiotherapy. Nat Rev
Cancer 2003;3(2):117–129. [PubMed: 12563311]
Regulation of autophagy by cytoplasmic p53
- E Tasdemir
- M C Maiuri
- L Galluzzi
- I Vitale
- M Djavaheri-Mergny
- D 'amelio
Tasdemir E, Maiuri MC, Galluzzi L, Vitale I, Djavaheri-Mergny M, D'Amelio M, et al. Regulation
of autophagy by cytoplasmic p53. Nat Cell Biol 2008;10(6):676-687. [PubMed: 18454141]
Transgenic mice with p53-responsive lacZ: p53 activity varies dramatically during normal development and determines radiation and drug sensitivity in vivo
- Komarova
Suppression of apoptosis in the PKCdelta null mouse in vivo
- Humphries