BMC Cancer 2007, 7:95 http://www.biomedcentral.com/1471-2407/7/95 Introduction Adenoid cystic carcinoma of salivary glands (ACCs) is a high malignant carcinoma characterized by intensive local invasion and insidious distant metastasis to the lung at an early stage, which is responsible for a poor long-term survival rate [1]. Although reasons of the invasiveness and aggressive metastatic dissemination of ACCs remain unclear, angiogenesis might be a possible involving mech- anism [2]. Our previous study showed that the ACCs his- tological type which presented high metastasis tendency exhibited higher microvessel density levels and more intensive expression of angiogenic related factors [3]. Angiogenesis, the development of new blood vessels from the pre-existing vascular beds, is an essential pathophysi- ologic event occurring in tumor growth and metastasis. There are various factors involved in angiogenesis, includ- ing the vascular endothelial growth factor (VEGF), which plays a key role in regulating tumor vascularization [4]. In addition, VEGF has demonstrated a major association with initiating the process of angiogenesis through regu- lating proliferation, migration, and differentiation of endothelial cells [5]. On the other hand, the nuclear tran- scription factor κB (NF-κB) that was reported increased in cancers and promoted tumor angiogenesis [6]. Once acti- vated, NF-κB p50 and p65 will translocate into nuclear and up-regulates a number of genes necessary for the ang- iogenesis of tumors, which have κB binding sites in their promoter regions [7]. Further, recent evidence has indi- cated that an over-expression of NF-κB is the key compo- nents of the angiogenic cascade, which contribute to VEGF-induced angiogenesis through up-regulation of VEGF mRNA expression in many tumors [5,8]. Our previ- ous study has demonstrated the expressed relationship between VEGF and NF-κB: the staining intensity of VEGF was significantly correlated with NF-κB nuclear localiza- tion rate in 80 ACCs clinical samples [3]. To our knowledge, however, there were few reports about the relationship between the distant metastasis character and the angiogenesis ability in ACCs, mainly because it is uncommon, and more than a decade of observation might be required to appreciate the prolonged clinical course in some patients. As a result, using an in vitro model to study the angiogenesis ability in high and low metastasis ACCs cell lines is necessary. The low metastasis cell line ACC-2 was established from ACCs in 1988 [9], and the high metastasis cell line ACC-M was a highly metastastic clone to the lung selected from ACC-2 [10]. The metastatic rate was 96% vs. 18% for ACC-M and ACC- 2 cell line. Since the high and low metastasis cell lines were established, reports have been made to explore the difference between the two cell lines on the cell and molecular level associated with tumor metastasis [11]. In our present research, the relationship between angio- genesis and metastasis in ACC-M and ACC-2 cell lines has been studied. We use the in vitro angiogenesis model to compare the angiogenesis abilities in high and low metas- tasis cell lines of human ACCs. Furthermore, the NF- κBp65 activity and VEGF expression levels in the two cell lines were also detected. Methods Cell culture The high and low metastasis cell lines of human ACCs (ACC-2 and ACC-M) [12] were obtained from the China Center for Type Culture Collection. The umbilical vein endothelial cell line (UVEC) was from the Key Lab for Oral Biomedical Engineering of Ministry of Education at Wuhan University. Cells were maintained at 37°C in DMEM and supplemented with 10% fetal bovine serum (GIBCO, Trace Biosciences Ltd., Sydney, Australia) under 5% CO2/95% air atmosphere and passed at a 4–7 split before use in the following experiments. To obtain the conditioned medium (CM), sub-confluent ACC-M and ACC-2 cells were serum starved for 12 h in 6 well plates (Greiner, Labotechnik, Germany). The medium was replaced with 1.0 ml serum-free DMEM medium, and the cells were then incubated for 12 h, after which the CM were obtained, stored at -20°C, and used for the following experiments. The viability of cells was estimated by CCK-8 kit (Japan, Kumamoto, Dojindo). Electrophoretic Mobility Shift Assay EMSA were performed using nuclear extracts as follows: 10 ug of nuclear extract in a 10-ul reaction volume was incubated on ice for 40 min. Double-stranded Oligonu- cleotide DNA probes, (κB: 5'-AGTTGAGGGGACTTTC- CCAGGC-3', and Oct-1:5'-TGTCGAATGCAAAT CACTAGAA-3') were end-labeled with 32P-γ, and applied to a 4% nondenatured polyacrylamide gel. Equal loading of nuclear extracts was confirmed by determining Oct-1 DNA binding activity. After electrophoresis, the gel was dried for 1 h at 80°C and exposed to Kodak X-ray film (Eastman Kodak Co., Rochester, NY, USA) at -80°C. Semi- quantitative Reverse Transcription-PCR Total RNA was extracted from 1 × 106 of ACC-M and ACC- 2 cells using TRIzol (Invitrogen, crop. Carlsbad, CA, USA). Aliquots (1 ug) of RNA were reverse transcribed to cDNA and aliquots (4 ul) of cDNA were used as a template for PCR using a PE9700 RT-PCR system (Applied Biosystems, Singapore) according to the manufacturer's instructions. The primers sets were as follows: VEGF (682 bp), 5'ggc tct aga tcg ggc ctc cga aac cat3' and 5'ggc tct aga gcg cag agt ctc ctc ttc3'; β-actin(434 bp), 5'tgt gcc cat cta cga ggg gta tgc3' and 5'ggt aca tgg tgg tgc cgc cag aca3'. Thermocycling con- ditions were melting at 95°C for 30 s; anneal at 63.5°C Page 2 of 7 (page number not for citation purposes)