Zhu et al. Journal of Experimental & Clinical Cancer Research 2012, 31:31 http://www.jeccr.com/content/31/1/31 Page 3 of 7 NT_009952.14, -1082 to +118), was used to prepare the reporter constructs. DNA was extracted from MDA- MB-435 cells. BLyS promoter was amplified by PCR using following primers: 5’- GCG GTA CCA AGC CTG GGT CTG GAG TTC T-3’ (forward) and 5’- GCC TCG AGC CTT TCT GCC TTT CTG CAT C-3’ (reserve). Cloned fragments were recovered and ligated into pGL3-basic luciferase vector. DNA transfectants were prepared using QIAprep spin miniprep kit. Cells were cultured in 24-well plates to 70-80% of confluence, and then transfected with 1 μg of pGL3-Basic/BP or pGL3- Basic. Plasmid pRL-SV40 Renilla luciferase reporter (20 ng) was used as internal control. Supernatant was removed after 24 h and the cells were subsequently trea- ted with CAPE for 12 h. Cell extracts were prepared and analyzed for luciferase activity using Dual-luciferase reporter assay system. Luciferase activity was expressed as relative luciferase activity (RLA). Statistical analyses The results are presented as the mean ± SD where applicable. Data were analyzed using GraphPad Prism 5.0 and the Student’s t-test to determine the level of sig- nificance. Statistical difference was accepted at p < 0.05. (GraphPad Prism 5.0 was used to perform statistical analysis.) Results and discussion Expressions of BLyS, TACI, BCMA and BAFF-R in human breast cancer cell lines The expression of BLyS has been detected in monocytes, macrophages and T cells. The three receptors are mainly in B cells, T cells and several kinds of malignant cells [10]. It is reported that both BLyS and its receptors are present in Ramos cells [11,12]. As shown in Figure 1A, BLyS and the receptor proteins were present in MDA-MB-435, MDA-MB-231 and MDA-MB-468 cells by immunofluorescence and Western Blotting. Ramos cells were used as positive control. However, BAFF-R chiefly accumulated in the nucleus of MDA-MB-435 and MDA-MB-231 cells, indicating that BAFF-R may act as a transcription regulator of certain target genes including BLyS, CD154 and so on. It is reported that BAFF-R is capable of functioning both as a growth/sur- vival cell membrane receptor, as well as a transcription factor or cofactor to promote B-cell survival and prolif- eration [13]. Further studies are necessary for confirm- ing this hypothesis. As shown in Figure 1B, the mRNA level of BLyS in MDA-MB-435 cell was dramatically increased in hypoxic conditions based on Q-PCR assay. In Figure 1C, protein level of BLyS was significantly elevated in hypoxic conditions for 3 h to 6 h. On the basis of Wes- tern Blotting data in MDA-MB-435 cells, we observed that BLyS was present not only as a dimer (~32 kDa) in plasma membrane and cytoplasm, but also as a trimer (~52 kDa) in supernatant. Both of the BLyS signals (~32 kDa and ~52 kDa) were strongly enhanced by the low oxygen tension. Migration of human breast cancer cells in the presence of BLyS We determine breast cancer cells migration when trea- ted with BLyS in both normoxic and hypoxic conditions. As seen in Figure 2, BLyS significantly enhanced the migration of MDA-MB-435, MDA-MB-231 and MDA- MB-468 cells in vitro compared with the negative con- trol. The responses of the three cell lines to BLyS were different. BLyS treatment caused dose-dependent response in MDA-MB-435 and MDA-MB-468. How- ever, no difference was found between the migration of MDA-MB-231 when treated with 10 ng/ml of BLyS compared to 0.1 ng/ml or 1 ng/ml of BLyS. Role of p65 activation in BLyS up-regulation NF-kappa B is critical for the regulation of apoptosis, viral replication, tumorigenesis, inflammation and var- ious autoimmune diseases. It is activated by a variety of stimuli such as hypoxia [14]. We also explored the pos- sible involvement of HIF-1a which can be modulated by low oxygen tension in cells and tissues. HIF-1a leads to the transcriptional induction of a series of genes that participates in angiogenesis, iron metabolism, and glu- cose metabolism [15]. HIF-1a was up-regulated and p65 was translocated by hypoxia (Figure 3A). CAPE, a NF-kappa B antagonist, specifically inhibits NF-kappa B activation and PX 12 attenuates expressions of HIF-1a and VEGF. Decreased activation of p65 resulted in BLyS downregulation in MDA-MB-435 cells (Figure 3B). MDA-MB-435 cells were transfected with pGL3-Basic/BP plasmid and then treated with CAPE or PX 12 for 12 h. The RLA data suggested that CAPE rather than PX-12 decreased the BLyS promoter activity significantly (Figure 3C). Immu- nofluorescence showed that p65 could be activated by hypoxia and CAPE was against the activation. It also showed that CAPE blocked expression of BLyS in hypoxic conditions (Figure 3D). The preceding results showed that translocation of p65, rather than accumula- tion of HIF-1a, was responsible for BLyS up-regulation. Activation of akt protein involved in BLyS-enhanced cell migration We have found that BLyS stimulated human breast can- cer cell migration. Activation of Akt and p38 MAPK pathways might contribute to BLyS-enhanced cell migration. SB 202190 is a p38 MAPK antagonist and API-1 is an Akt/protein kinase B (PKB) antagonist.