Biological effects of epigallocatechin gallate during the protection of the submandibular gland after injury

Abstract

Background: When radiotherapy is delivered to head and neck cancer (HNC) patients, the salivary gland (SG) secretory epithelia can be irreversibly injured in up to 60% of the individuals, leading to dry mouth or xerostomia. Radiotherapy’s effectiveness in suppressing HNC growth is correlated with an increase in free radicals that produce DNA damage to the tumor and neighboring organs like the SG. Epigallocatechin gallate (EGCG) is one of the most abundant polyphenols present in green tea leaves and a well-known antioxidant. In previous in vitro study using genetically modified immortal SG cell lines, EGCG protected SG cells from γ-radiation. However, the ability of EGCG to maintain SG epithelia during homeostasis and to provide radioprotection for SG organ is poorly understood, and thus it requires further investigations.

Aim: To investigate whether EGCG supports epithelial maintenance during salivary gland homeostasis and determine if EGCG protects the salivary gland from epithelial injury induced by radiotherapy.

Methods: In the homeostasis SG developmental model, ex vivo fetal mouse submandibular glands were cultured with EGCG for 72h at 7.5-30 µg/mL. Next, SG epithelial branching morphogenesis was measured by bright-field microscopy and gene expression arrays. In the injury SG model, conventional linear accelerator (LINAC) technology for radiotherapy was used at 5-10 Gy to determine the optimal dose for generating radiation injury. To confer EGCG protection, glands were pretreated with EGCG at 7.5-15 µg/mL for 24 hours and induced by 7 Gy and then compared to the irradiated group after cultured for 48h. To measure the end bud growth, epithelial growth quantification using bright-field microscopy was performed every 24h. Laser confocal scanning microscopy, gene expression arrays, the Griess assay, and whole-mount immunohistochemistry (IHC) were used to evaluate the biological effects of EGCG on SG epithelial cells.

Results: In ex vivo SG organ culture conditions, EGCG at 7.5 µg/mL maintained epithelial SG homeostasis during development. After radiation injury, EGCG pre-treatment protected the growth, mitosis, and maturation of the epithelia, generated a mature SG epithelial acinar and ductal compartment, increased the epithelial stem cell niche (Sox2+), decreased radiation-induced cellular apoptosis, and reduced the oxidant stress markers.

Benefit: This research work led to a better understanding of the therapeutic potential of EGCG to prevent radiation-induced epithelial SG injury in the ex vivo fetal organ.