Sympathetic Neurotransmitter NE Contributes To The Perineural Invasion Of Human Salivary Adenoid Cystic Carcinoma

Salivary adenoid cystic carcinoma(SACC) is a common malignancy which occurs in salivary gland, its incidence ranks second among the malignancies of salivary gland. SACC is notorious for its perineural invasion(PNI) feature. PNI is defined by tumor’s invasion of nerves of adjacent tissue, which results in difficult clinical diagnosis and treatment of SACC. Norepinephrine(NE) is a major component of the sympathetic response, it is manufactured by nerves of the sympathetic nervous system. NE is reported to accelerate the progression of tumors through the activation of β2-adrenergic receptors(β2-ARs). Thus, it is important to study the roles of NE and β2-AR in the PNI of SACC.Objective:1. To investigate the expression of sympathetic nerve and β2-AR in SACC and normal salivary gland.2. To investigate the expression of β2-AR in cell lines SACC-83、SACC-LM and SCC-25.3. To investigate the influence of sympathetic neurotransmitter NE on the proliferation, migration and invasion of SACC-83, SACC-LM, SCC-25 and Fa Du cell lines. And investigate the influence of pretreatment with nonselective β-adrenergic blocker propranolol or β2-adrenergic receptor blocker ICI118,551 on the effects of NE.4. To investigate the influence of NE on the perineural invasion of SACC-83 and SACC-LM cell lines. And investigate the influence of pretreatment with propranolol or ICI118,551 on the effects of NE.Methods:1. Using immunohistochemistry staining to study the expression of sympathetic nerve and β2-AR on tissue sections from SACC(18 cases) and normal salivary gland(10 cases). Analyse the possible influence of sympathetic nerve and β2-adrenergic receptor expression on PNI of SACC.2. Using immunocytochemistry staining and Western blot to study the expression of β2-AR on SACC-83, SACC-LM and SCC-25 cell lines.3. Using MTT cell activity detection and Transwell chamber cell migration and invasion assay to prove the impact of NE, propranolol or ICI118,551 on the cell viability, migration and invasion ability of SACC-83, SACC-LM, SCC-25 and Fa Du cell lines.4. Using in vitro perineural invasion assay to prove the impact of NE, propranolol or ICI118,551 on the perineural invasion ability of SACC-83 and SACC-LM cell lines.Results:1. The positive rate of sympathetic nerves was 72.2%(13/18)in SACC. In SACC, sympathetic nerves were mainly distributed in the tumor stroma, a few nerves were wrapped by tumor parenchyma, and the manifestation of sympathetic nerves was relatively coarse. In normal salivary gland, sympathetic nerve fibers were broadly and sparsely distributed in glandular tissues, they seemed like small dots or fine cords.2. β2-ARs were found in SACC and normal salivary gland, respectively. In SACC, β2-ARs were widely distributed on the cell membranes of tumor cells, but not all of the tumor cells’ membranes were colored. In normal salivary gland, β2-ARs were widely expressed on membrane or cytoplasm of tumor cells, and gland duct cells were strongly stained.3. Both immunocytochemistry staining method and Western blot proved that β2-ARs were expressed in SACC-83, SACC-LM and SCC-25 cell lines. The expression of β2-ARs in SCC25 cell line was the strongest, while SACC-83 was weaker, and SCC-25 was the lowest.4. A range of concentrations of NE can promote the proliferation, migration and invasion of SACC-83, SACC-LM, SCC-25 and Fa Du cell lines. However, the promotion effects of NE can be inhibited by pretreatment of cell lines with propranolol or ICI118,551.5. 10 μmol/L NE can promote the perineural invasion of SACC-83 and SACC-LM cell lines. However, the promotion effects of NE can be inhibited by pretreatment of cell lines with propranolol or ICI118,551.Conclusion:1. The expression of sympathetic nerve or β2-ARs between SACC and normal salivary gland were different. The difference indicates that sympathetic nerve may influence PNI of SACC through releasing norepinephrine to bind β2-ARs.2. β2-ARs were expressed on SACC-83, SACC-LM and SCC-25 cell lines, and the cell lines can be used as in vitro experiment models to study SACC and OSCC.3. The promotion effects of NE can be inhibited by pretreatment of SACC-83, SACC-LM, SCC-25 and Fa Du cell lines with propranolol or ICI118,551, which indicates that sympathetic transmitter norepinephrine may influence the proliferation, migration and invasion of SACC and OSCC via β2-ARs.4. The promotion effects of NE can be inhibited by pretreatment of SACC-83 and SACC-LM cell lines with propranolol or ICI118,551, which indicates that sympathetic transmitter norepinephrine may influence the perineural invasion of SACC via β2-ARs.In this article, we mainly focused on how sympathetic nerve and sympathetic neurotransimitter influence the PNI of SACC. In addition, the impact of parasympathetic nerve and parasympathetic neurotransmitter on the PNI of SACC is in progress.