Radiation-Induced Salivary Gland Dysfunction: Mechanisms, Therapeutics and Future Directions

Abstract

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.

Keywords: P2 receptors; bystander effect; head and neck cancer; hyposalivation; purinergic signaling; radiation; radioprotection; saliva; salivary gland; xerostomia.

Figure 1

Timeline of Radiation-Induced Changes in the Rodent Salivary Gland. Following irradiation, rodent models show decreased saliva flow at approximately 3 days and a loss of amylase secretion reported as early as 4 days in rats post-IR [2,56,58]. In the acute phase, immediate DNA damage [6,59], rapid apoptosis of acinar cells [4,6,58], and elevated levels of intracellular calcium [45,46] and reactive oxygen species [45,46,60,61] contribute to acute loss of glandular function following irradiation. This period is also marked by release of ATP, which activates the P2X7 receptor (P2X7R), and P2X7R-dependent release of prostaglandin E₂ (PGE₂) in murine parotid cells [48]. During the transition phase, loss of apical/basolateral polarity as a result of PKCζ inactivation [55,62,63], increases nuclear Yes-associated protein (Yap) levels [55,64], compensatory proliferation [62,65,66], cellular senescence [60,67,68], and cytoskeletal rearrangements [50,69], which contribute to long-term dysfunction. Changes in innervation and vasculature have been reported as early as 24 h post-IR [53], as well as at chronic time points [54,70]. Though inconsistently reported, fibrosis generally appears between 4 and 6 months following irradiation [1,44,71]. There is little information regarding the effect of irradiation on the immune landscape of the salivary glands in rodent models, although one study indicates changes at 300 days post-IR in mice [54].

Figure 2

Radiation-Induced Bystander Effects. The bystander effect can be propagated by intercellular communication between adjacent cells via gap junctions or by autocrine or paracrine signaling processes whereby NTPs, nitric oxide (NO), reactive oxygen species (ROS), cytokines (e.g., TGF-β), or other second messengers elicit a response in non-irradiated cells. Created with Biorender.com.

Figure 3

Purinergic Signaling in Bystander Effects. In response to elevated extracellular ATP (eATP) levels following irradiation, P2Y₂R and P2X7R, which are expressed in murine and human salivary glands, may mediate a number of bystander effects. Through activation of the NLRP3 inflammasome, P2X7Rs promote ROS production, growth factor maturation (e.g., IL-1β) and subsequent release and apoptosis. Activation of either P2Y₂R or P2X7R causes increased [Ca²⁺]_i and subsequent downstream signaling processes (e.g., ERK1/2 signaling, gene expression changes, inflammatory processes). P2Y₂R activates phospholipase C (PLC) resulting in the production of inositol 1,4,5 trisphosphate (IP₃) and diacylglycerol (DAG), in turn mobilizing intracellular Ca²⁺ and activating protein kinase C (PKC), respectively, and subsequent downstream signaling. Through its C-terminal Src-homology 3 (SH3) domain, the P2Y₂R is involved in Src-dependent activation of growth factor receptors (e.g., EGFR, VEGFR-2) and downstream MAPK signaling as well as transactivation of EGFRs through activation of metalloproteases, ADAM10 and ADAM17. P2Y₂Rs may also contribute to the bystander effect by promoting latent TGF-β signaling. DAG: diacylglycerol; PLC: phospholipase C; PKC: protein kinase C; ERK: extracellular signal-regulated protein kinase; JNK: c-Jun N-terminal kinase; MAPK: mitogen-activated protein kinase; COX: cyclooxygenase; PGE₂: prostaglandin E₂; ADAM: A Disintegrin And Metalloprotease. Created with Biorender.com.

Figure 4

Pharmacological Approaches to Salivary Gland Radioprotection and Regeneration. Amifostine is currently the only radioprotective therapeutic approved for the prevention of RT-induced hyposalivation. The membrane-bound alkaline phosphatase converts Amifostine to WR-1065 that is then taken up by the cell. WR-1065 is thought to promote radioprotection by scavenging reactive species in turn affecting gene expression, apoptosis, chromatin stability, DNA damage repair and enzymatic activity [221,222]. Other promising radioprotective therapeutics being investigated in preclinical animal models include the P2X7R antagonist, A-438079 [48], and the tyrosine kinase inhibitors, dasatinib and imatinib [77,78]. Pharmacological approaches to regeneration studied in animal models post-IR target a number of signaling pathways. IGF-1 treatment 4–7 days post-IR restored saliva production in a PKCζ-dependent manner [55]. mTOR signaling is another target that has been investigated by several groups to promote salivary gland regeneration [88,223]. Administration of the rapamycin analog, CCI-779, following IR improved saliva flow rates at 30 days post-IR [88]. Transient upregulation of Shh signaling by either overexpressing a Shh transgene or by administering a smoothened agonist, restored stimulated saliva flow [224]. EDAR agonists, such as monoclonal antibodies that promote EDAR signaling, are essential for salivary gland development and have shown promise in restoring salivary gland function in mice [65]. The senolytic agent, ABT263, which depletes senescent cells by inhibiting BCL-2 and BCL-xL, has been shown to promote salivary gland regeneration and self-renewal capabilities of residual salivary gland stem cells [68]. RTK: receptor tyrosine kinase; PLC: phospholipase C; PKC: protein kinase C; BCL: B-cell lymphoma; TRAF: tumor necrosis factor receptor-associated factor; IKK: IkB kinase; AKT: protein kinase B; EDAR: ectodysplasin A-1 receptor; PTCH: patched receptor. Created with Biorender.com.