Jang et al. BMC Cancer 2010, 10:391 http://www.biomedcentral.com/1471-2407/10/391 not degradated at 24 hours of ANT2 shRNA transfec- tion in contrast to the marked decrease of Akt phos- phorylation. Taken together, these findings suggested that an inhibitory effect of ANT2 shRNA on the PI3K/ Akt signaling pathway in HER2/neu-overexpressing breast cancer cells (SK-BR3) might be mediated by degradation of HER2/neu protein rather than Akt down-regulation, especially in the early time of transfection. ANT2 shRNA suppresses VEGF expression, probably through inhibition of PI3K/Akt signaling pathway Here, we examined whether ANT2 knockdown by shRNA affected expression of VEGF, and whether this might be regulated by the PI3K/Akt signaling pathway. In cancer progression, PI3K/Akt and NF- B signaling pathways are strongly associated with growth, migration, invasion, angiogenesis, and metastasis of tumor. PI3K plays a central role in a diverse range of cellular responses, including cell growth and survival. Akt (also named protein kinase B) is a downstream target of PI3K and is also a critical mediator of survival signals for pro- tection of cells from apoptosis. VEGF, induces modifica- tion of the actin cytoskeleton and influences endothelial cell permeability and migration during angiogenesis. Here, we examined the question of whether or not ANT2 knockdown by shRNA affected on the VEGF expression, and if which might be regulated by PI3K/Akt signaling pathway. ANT2 shRNA transfection markedly decreased VEGF mRNA level, and these changes occurred in con- cert with changes in VEGF protein levels (Fig. 3A). As expected, suppression of VEGF expression by ANT2 shRNA seemed to be dependent on ANT2 shRNA- induced inhibition of PI3K/Akt signaling, considering that PI3K inhibitor, LY294002, dramatically down-regu- lated VEGF expression in SK-BR3 cells (Fig. 3B). ANT2 shRNA down-regulates MT1-MMP and inhibits expression and activity of MMP2 and MMP9 by inhibiting PI3K/Akt signaling pathway A decrease in VEGF protein suggested a concurrent decrease in MT1-MMP protein, because MT1-MMP has previously been known as a downstream target of VEGF. In the present study, we found that MT1-MMP was significantly decreased in ANT2 shRNA transfected SK-BR3 cells in both mRNA and protein levels (Fig. 4A). Furthermore, this inhibition of MT1-MMP by ANT2 shRNA was interrupted and the expression of MT1-MMP was restored by wild-type PI3K overexpres- sion (Fig. 4B). These results suggested that inhibition of PI3K/Akt signaling by ANT2 shRNA might play an important role in the down-regulation of MT1-MMP Transfection with ANT2 shRNA was found to signifi- cantly decrease expression of both MMP2 and MMP9 Page 5 of 9 Figure 3 ANT2 shRNA down-regulates VEGF expression, which is dependent on the PI3K signaling pathway. (A) Cells were transfected with scramble shRNA or ANT2 shRNA and cultured for 24 hours. Total RNA was extracted from respective cell lines and subjected to RT-PCR using specific primers for human VEGF or GAPDH (internal control). Intracellular levels of VEGF were also detected by flow cytometry. (B) Cells were pre-treated with PI3K inhibitor, LY294002. After 2 hours of incubation, cells were transfected with scramble shRNA, ANT2 shRNA, pcDNA, or pcDNA- ANT2 and cultured for 24 hours. Total RNA was extracted from respective cell lines and subjected to RT-PCR using specific primers for human VEGF or GAPDH (internal control). (Fig. 5A). To determine whether or not activity of MMP2 and MMP9 is also inhibited by ANT2 shRNA, gelatin zymography analysis was performed, which showed that activation of pro-matrix MMP2 and pro- matrix MMP9 was markedly suppressed in the superna- tants of SK-BR3 cells transfected by ANT2 shRNA (Fig. 5B). These findings suggested that ANT2 shRNA sup- pressed MMP2 and MMP9 expression and activity in HER2/neu-overexpressing breast cancer cells. ANT2 shRNA suppresses invasiveness and migration of HER2/neu-overexpressing breast cancer cells Based on the above observation, we finally studied the effects of ANT2 shRNA on invasion and migration of breast cancer cells. In vitro matrigel invasion assay and transwell migration assay showed that ANT2 shRNA significantly suppressed the invasion and migration of SK-BR3 cells after 24 hours of transfection (Fig. 6). At that time, ANT2 shRNA had little influence on the cell viability by MTT assay, although the morphology