BMC Cancer 2007, 7:95 http://www.biomedcentral.com/1471-2407/7/95 uncompleted tubule structures on the surface of gel, and almost no sprouting cells could be observed. Discussion The great potential for hematogenous metastasis at an early stage is one of the unique characters of ACCs. Our previous study showed that the high expressions of angio- genesis related factors NF-κB and VEGF were significantly correlated to lung metastasis and solid histotype, which present high metastasis tendency [3]. In the current study, we used high and low metastasis cell lines of human ACCs-ACC-M and ACC-2- to compare the angiogenic related factor NF-κB and VEGF expression levels. We also compared the ability of angiogenesis between the two cell lines by in vitro cell migration, tube formation and sprouting assay. The tumor cells induced secretion of angiogenesis factors which is commonly observed in most aggressive tumors [16]. Among various angiogenic factors, the most notable is VEGF, which exerts its mitogenic activity especially on endothelial cells [5]. Our findings revealed that NF-κBp65 activity detected in the high metastasis cell line ACC-M was much greater than that in the low metastasis cell line ACC-2, and the VEGF mRNA expression level in the high metastasis cell line ACC-M was almost 2-fold than that in the low metastasis cell line ACC-2. Moreover, the protein staining intensity of VEGF in ACC-M is also significantly higher than that in ACC-2. The results are consistent with the evidence that the tumor-induced VEGF expression and NF-κB activity correlated with tumor metastasis [8,17]. It was suggested that the aberrant activity of NF-κB and VEGF level might be the possible mechanisms involved in the high metastasis ability of ACC-M cell lines. In the neovascularization of several neoplasms, research have shown the potent angiogenic factor VEGF, whose genes have a κB binding site, are regulated by activated NF-κB [18,19]. Once triggered and activated, freed NF-κB, in the heterodimer of the p65 and p50 subunits, translo- cates from the cytoplasm into the nucleus and binds to the specific sequence in the promoter of target genes. In our study, it was found that the constitutive activity of NF- κBp65 detected in ACC-M and ACC-2 cells. In these cell lines, inhibition of NF-κBp65 activity by a PCMV vector mediated expression of phosphorylation defective mutant of IκBα effectively inhibited the expression of VEGF mRNA. Furthermore, the nuclear staining rate of NF- κBp65 was significantly correlated with VEGF protein level in both ACC-M and ACC-2 cell lines. These results suggest that NF-κBp65 is involved in the regulation of VEGF expression. This hypothesis is supported by the findings of others. The study of Tamami et al. [20] dem- onstrated the inhibitors of the transcription factors NF-κB completely prevented the advanced glycation end prod- ucts (AGE)-induced up-regulation of VEGF mRNAs and the subsequent increase in DNA synthesis in endothelial cells. The authors suggested that NF-κB activation might be involved in the AGE-elicited angiogenesis through overproduction of auto-secretion VEGF proteins. Huang et al. demonstrated a concordant increase in NF-κB activ- ity with the elevated VEGF mRNA in ovarian cancer cells [21]. In this cell line, stable expression of mutated IκBα resistant to degradation, decreased NF-κB activity and reduced VEGF mRNA expression, suggesting that the reg- ulation of VEGF by NF-κB is mediated at the transcription level. The angiogenic process includes endothelial cell activa- tion, proliferation, migration, tube formation, and capil- lary sprouting [22]. It increases the opportunity to improve the development of metastases [23] in many malignant tumors, such as gastro-intestinal tumors [24], colorectal cancer [25], lung adenocarcinoma [26], and hepatocellular carcinoma [27]. VEGF and NF-κB have been reported to be crucial in new blood vessel formation [14]. Those angiogenic-related factors increase the oppor- tunity for malignant cells distance metastases through the leakage basement vessel, and hence profoundly influence the prognosis of cancer patients [5,17]. To further indicate the angiogenesis abilities in different metastasis cells lines of ACCs, the in vitro angiogenesis model was also employed. Through in vitro three-dimensional ang- iogenic assay model, our present study showed ACC-M, the highly metastasis cell clone, could remarkably enhanced the migration and tube formation of UVECs and induce UVECs sprouting into the gel matrix. ACC-M cells presented higher angiogenic activity might result from its higher VEGF expression levels and NF-κB activity which were crucial in tumor angiogenesis [8,20,21]. The findings of Ishibashi's experiment also showed that spe- cific VEGF antibody could inhibit the in vitro angiogenic activity in human salivary gland carcinoma cells [2]. As a result, it might raise the possibility that the high metasta- sis cell line ACC-M present higher angiogenic-related fac- tors might facilitate the further angiogenesis. In conclusion, our current study has shown human ACCs cells with high metastasis potential express high levels of constitutive NF-κB p65 activity and VEGF expression. Sup- pression of NF-κB p65 activity through stable expression of a phosphorylation defective IκBα mutant (S32, 36A) significantly decreased VEGF expression. Furthermore, ACC-M could remarkably stimulate the migration and tube formation of endothelial cells and induce UVECs sprouting into the gel matrix. The results indicate that in ACCs, cells with higher metastasis potential might present greater angiogenesis ability. Our further study will investi- gate the function of NF-κB p65 signaling in angiogenesis and metastasis of ACCs cells. Page 6 of 7 (page number not for citation purposes)