Study On The Mechanisms Of Neural Invasion In Adenoid Cystic Carcinoma Of Lacrimal Gland

Objective1. To detect the expression and correlation of GDNF, GFRa-1, Ret, NCAM and Survivin in adenoid cystic carcinoma of lacrimal gland as well as their relations with perineural invasion of adenoid cystic carcinoma(ACC) of lacrimal gland.2. To study the effect of GDNF on ACC-2cells proliferation and migration, and to provide experimental evidence for the mechanisms of perineural invasion in adenoid cystic carcinoma of lacrimal gland.3. To investigate the relations of protein and mRNA of GDNF, GFRa-1, NCAM and Survivin in ACC-2cells respectively, and we would grasp their interaction and association in the microenvironment of ACC to provide references for clinically effective targets.Methods1. The expression of GDNF, GFRa-1, Ret, NCAM and Survivin was evaluated in the51operative samples of adenoid cystic carcinoma of lacrimal gland as well as5samples of normal lacrimal gland by SABC immunohistochemical staining.2. MTT assay, the growth curve, flow cytometry was employed to observe the influence on the proliferation and apoptosis of ACC-2cells with different concentrations (time) courses of GDNF. Meanwhile, Transwell chamber was used to observe the migration effect of GDNF on ACC-2cells.3. When exposed to determinate concentrations of GDNF, protein and mRNA of GDNF, GFRa-1, NCAM and Survivin in ACC-2cells were detected by Western blotting and RT-PCR respectively.Results1. Expression rate of GDNF, GFRa-1, Ret, NCAM and Survivin in the samples of adenoid cystic carcinoma(ACC) of lacrimal gland was as following:62.7%(32/51),62.7%(32/51),54.9%(28/51),60.8%(31/51),60.8%(31/51)each of which was higher than in normal lacrimal gland tissue. The perineural invasion(PNI) rate of solid pattern ACC and recurrent tumor group were higher than in adenoid-tubular pattern ACC and primary tumor group, there were significant differences between two groups (P<0.05), and there were no significant differences between pain group and no-pain group (P>0.05).2. Expression rate of NCAM and Survivin in solid pattern ACC was higher than in adenoid-tubular pattern ACC, Expression rate of GFRα-1, Ret, NCAM and Survivin in recurrent tumor group was higher than in primary tumor group, Expression rate of GDNF, GFRa-1, Ret and NCAM in PNI tumor group was higher than in no-PNI tumor group, there were significant differences in above-mentioned groups(P<0.05).3. There was significant correlation between the expression rate of GDNF and GFRa-1, Ret, NCAM in ACC of lacrimal gland, but no significant correlation between the expression rate of GDNF and Survivin.4. With standard cultivated for48hours, the proliferation of ACC-2cells was stimulated when exposed to incremental concentrations of GDNF(20～120ng/ml), which had statistical significant differences compared with control group. The effect of GDNF on ACC-2cells proliferation reached its maximum at the concentration of80ng/ml. On the72th hour, GDNF(80ng/ml) can promote the proliferation of ACC-2cells significantly which got the coincidental tendency to the growth curve interfered in GDNF(80ng/ml).5. After standard cultivated for48hours with GDNF(80ng/ml), the ratio of ACC-2cells in G1period was decreased compared with control group, and the ratio of ACC-2cells in S period was increased by flow cytometry.6. With standard cultivated for40hours, the count of migration cells was increased by GDNF(60-160ng/ml). The effect of GDNF on ACC-2cells migration reached its maximum at the concentration of160ng/ml. From the24th hour, GDNF (100ng/ml) can promote the migration of ACC-2cells in a time-dependent manner(24h,30h,40h), which had statistical significant differences compared with control group(P<0.05).7. GDNF(80ng/ml) enhanced the mRNA and protein expression of GFRα-1, NCAM, Survivin in ACC-2cells, and weakened the mRNA and protein expression of GDNF in ACC-2cells. Conclusions1. Aggressive biological behavior of ACC of lacrimal gland includes perineural invasion phenomenon, which can be used as a poor prognosis clinical observation of one of the indicators in the lacrimal gland ACC.2. GDNF, GFRa-1, Ret play an important role in the perineural invasion in ACC of lacrimal gland, which can become biological indicators to judge whether exists PNI phenomenon, and the latter two are expected to become one of the factors of prediction and assessment for prognosis.3. GDNF can promote cultured human ACC-2cells proliferation and migration, in a dose and time dependent manner respectively, but no significant role in regulation of GDNF on the level of ACC-2cells apoptosis.4. GDNF via GFRa-1-Ret signaling pathway, or by non-dependent Ret signaling pathway-NCAM, through the concentration gradient along the peripheral nerve-center of GDNF, as well as by virtue of NCAM different/same tropism adhesion, ACC and peripheral nerve attracted each other, forming a micro-environment conducive to PNI, and enhanced the ability of PNI for ACC cells.5. The expression of NCAM is associated with the pathological type of ACC of lacrimal gland, which become indicators for PNI and prognosis to ACC of lacrimal gland at the same time. Through the detection of NCAM expression around ACC of lacrimal gland, especially the expression in peripheral nerve nearby, you may find residual tumor cells or early PNI in ACC of lacrimal gland, and guide clinical treatment.6. The expression of Survivin is closely related to the pathological type and prognosis,of ACC of lacrimal gland but no obvious correlation with PNI of ACC of lacrimal gland, Survivin and GDNF may promote synergistic effect of ACC-2cells proliferation.