Nerve Growth Factor and Vascular Endothelial Growth Factor: Retrospective Analysis of 63 Patients with Salivary Adenoid Cystic Carcinoma

Aim To detect the expression of nerve growth factor （NGF） and vascular endothelial growth factor （VEGF） in salivary adenoid cystic carcinoma （SACC） tissues, as well as to determine the correlation between growth factor expression and prognosis in SACC. Methodology Medical records of 63 patients surgically treated for SACC between January 1988 and October 2005 were reviewed. Immunohistochemistry was performed to examine the expression of NGF and VEGF in tumor tissues. Kaplan-Meier analysis and Cox＇s proportional hazard regression model were applied to assess predictors of survival. Results NGF and VEGF were overexpressed in SACC tissues, compared with those in normal salivary tissues （P〈0.05）, and the staining intensity of these two factors was stronger in groups of solid subtype, advanced TNM stage, perineural invasion and recurrence. Patients with high- expression of NGF and VEGF, solid subtype, advanced stage, perineural invasion, recurrence and extended resection alone had worse survival rates （P〈0.05）. Conclusion NGF and VEGF are expressed increasingly in the tissues of SACC cases with invasion and metastasis. NGF expression and VEGF expression are independent prognosis factors for survival.

ANTIMETASTATIC EFFECT OF INTEGRIN IIb/IIIa INHIBITORSON SALIVARY ADENOID CYSTIC CARCINOMA

Objectives: To investigate the relation between metastatic potential of salivary adenoid cystic carcinoma (SACC) and tumor cell-platelet adhesion, and the antimetastatic effect of integrin IIb/IIIa inhibitor on SACC. Methods: Tumor cell-platelet adhesion of highly metastatic SACC-LM, non-highly metastatic SACC-83 and effect of aspirin, arginine-aspartate (RD), magnesium acetylsalicylate on adhesion were studiedin vitro. Antimetastafic effect of aspirin, RD, magnesium acetysalicylate on experimental metastasis of SACC was observedin vivo. Results: The tumor cell-platelet adhesion was stronger in SACC-LM than in SACC-83. Aspirin, RD and magnesium acetylsalicylate could inhibit the adhesion of SACC-LM at the concentration of 1, 5 and 25 μg/ml. RD can inhibit experimental metastasis of SACC. Conclusion: Metastasis of SACC is related to platelet-tumor cell adhesion, RD could inhibit metastasis of SACC.

EXPRESSION OF EPIDERMAL GROWTH FACTOR RECEPTOR IN DIFFERENT SALIVARY ADENOID CYSTIC CARCINOMA CELL LINES

Objective： To investigate the expression of epidermal growth factor receptor, a receptor tyrosine protein kinase, in the subcellular fractions of human salivary adenoid cystic carcinoma cell lines SACC-83 and SACC-LM. Methods： Low metastatic and high metastatic cells of the adenoid cystic carcinoma, SACC-83 and SACC-LM, were cultured. Their subcellular fractions were extracted. The expression of epidermal growth factor receptor was detected with Western blot method, and the results of protein expression were quantitatively analyzed by FluorChem V2.0 software. Results： The results of Western blot analysis indicated that, EGFR expression on the membrane of SACC-83 cells was significantly higher than that of SACC-LM cells, but its expression in cytoplasm was significantly less in the former than the later （P〈0.01）. In SACC-83 cell line, EGFR was over-expressed in membrane （P〈0.01）, but in SACC-LM cell line, EGFR was over-expressed in cytoplasm （P〈0.01）. Conclusion： The results suggest that the obtaining of metastasis ability is related to the high expression of EGFR protein in cytoplasm, so the molecular targeting therapy to EGFR may be an ideal treatment for the invasion and metastasis of salivary adenoid cystic carcinoma.

A20 inhibits human salivary adenoid cystic carcinoma cells invasion via blocking nuclear factor-kB activation

Background A20, also known as tumor necrosis factor α induced protein 3 （TNFaip3）, is a cytoplasmic zinc finger protein that inhibits nuclear factor kappa-B （NF-κB） activity and prevents tumor necrosis factor （TNF）-mediated programmed cell death. NF-κB is a transcription factor that regulates expression of genes involved in cell proliferation, cell survival and anti-apoptosis. Several studies have implicated that the NF-κB signal pathway is associated with angiogenesis and clinico-pathological process of adenoid cystic carcinoma （ACC） of the salivary glands.Methods The ability of overexpression of A2.0 to influence the biological behavior and invasion of ACC cells was examined. The cells were stably transfected with full-length A20 cDNA. Stable gene transfer was verified by realtime-polymerase chain reaction （PCR） and Western blot analysis. The change of cell biological behavior was examined by methyl thiazolyl tetrazolium （MTT） and NF-κB luciferase reporter assay and the invasion of the cells was examined by a Matrigel invasion chamber.Results pEGPFN3-A2.0 gene was stably transferred into ACC-2 cells and overexpressed. When cells were treated with TNFα, the NF-κB activity of ACC-2-A20 cells could be down-regulated about 46.32% in contrast to ACC-2-GFP cells （P〈0.05）. A20 potently inhibited growth of A20 transfectant ACC-2-A20 compared with control vector transfected groups and the ACC-2 empty control group （P〈0.05）. The ACC-2-A20 cells showed significantly reduced ability to invade through Matdgei-coated filters compared to ACC-2-GFP and ACC-2 cells. The inhibition rate was up to 71.05% （P〈0.05）. Conclusions A2.0 gene transfer is associated with decreased tumor invasion, in part via the down-regulation of NF-κB expression, providing evidence for a potential application of A20 in designing a treatment modality for salivary gland cancers such as ACC.

RNAi knockdown of C-erbB2 expression inhibits salivary gland adenoid cystic carcinoma SACC-83 cell growthin vitro

Objective：To knockdown the C-erbB2 gene in salivary gland adenoid cystic carcinoma SACC-83 cells using RNA interference,and determine the effect of silencing C-erbB2 on cell proliferation.Methods：C-erbB2-siRNA was transfected into SACC-83 cells.RT-PCR and immunohistochemistry were used to detect C-erbB2 expression in SACC-83 cells.Cell proliferation was measured by the MTT assay and gene knockdown was achieved by RNA interference.Apoptosis was analyzed by flow cytometry.Results：Compared with the control,C-erbB2 mRNA expression was decreased in the C-erbB2-siRNA transfection group,and immunohistochemical analysis indicated that C-erbB2 protein expression was decreased.After C-erbB2-siRNA was transfected for 48 h,absorbance at 570 nm （MTT）was 0.185±0.021 compared with 0.354±0.034,0.299±0.053,and 0.314±0.049 in the blank control,liposome control and negative control siRNA groups,respectively.The differences were statistically significant （P〈0.05）between the C-erbB2-siRNA group and the control groups.Following the C-erbB2 knockdown,the percentage of apoptotic cells was 5.63%compared with 2.04%,2.85%,and 2.98%in the three control groups,respectively.Proliferation of SACC-83 cells was inhibited,and early apoptotic cells were increased.Conclusion： RNA interference can effectively silence C-erbB2 gene expression and inhibit growth of SACC-83 cells,which indicates the potential of targeting this gene as a novel gene therapy approach for the treatment of salivary gland adenoid cystic carcinoma.

Inhibition of Salidroside on Salivary Gland Adenoid Cystic Carcinoma

To evaluate the role of salidroside on proliferation,apoptosis and invasiveness of salivary gland adenoid cystic carcinoma cells（SACC）,immunocytochemical staining was employed to detect proliferating cell nuclear antigen（PCNA）,caspase 3 and caspase 8 expression in SACC-2 cells.Modified Boyden chamber assay combined with laser confocal microscopy（LSCM） was used to evaluate the invasion and migration abilities of SACC-2 cells at different time point.Immunohistochemistry staining revealed that the expression of PCNA was significantly decreased（P0.01） after salidroside treatment.In contrast,salidroside treatment led to increased caspase 3 and caspase 8 in SACC-2 cells.Cell migration depth and number of cells that penetrated Boyden chamber were also decreased by salidroside.Salidroside potently inhibits the proliferation and simultaneously induces the apoptosis of SACC-2 cells.Migration and invasion of SACC-2 cells are also inhibited.Our data throw light on potential clinical application of salidroside to the patients with SACC.