Jang et al. BMC Cancer 2010, 10:391 http://www.biomedcentral.com/1471-2407/10/391 Page 6 of 9 Figure 4 ANT2 shRNA down-regulates MT1-MMP expression by suppressing PI3K signaling in SK-BR3 cells (A). Cells were transfected with scramble shRNA or ANT2 shRNA. After 24 hours of incubation, total RNA and cell extracts were prepared for RT-PCR using specific primers for human MT1-MMP or GAPDH (internal control) and western blotting with anti-MT1-MMP and anti-b-actin antibodies. (B) Cells were transfected with scramble shRNA or ANT2 shRNA with or without the wild-type PI3K expression vector and cultured for 24 hours. After 24 hours of incubation, total RNA and cell extracts were prepared for RT-PCR using specific primers for human MT1-MMP or GAPDH (internal control) and western blotting with anti-MT1-MMP and anti-b-actin antibodies. of cells seemed not to be the best under microscopic examination. We counted cells numbers in 10-indepen- det fields (5 mm × 5 mm) and averaged. These data altogether indicated that invasion (about 65% reduction) and migration (about 48% reduction) of breast cancer cells was suppressed or slowed by knockdown of ANT2 probably via inhibition of MMP2 and MMP9 expression and activity. Discussion Abnormal growth and metastasis of cancer cells are regarded as important biological characteristics of can- cer development and progression. Metastasis is the main cause of morbidity and mortality in millions of patients with cancer. During the complicated process of metasta- sis, invasion of cancer cells is the most important and characteristic step. Clearly, an agent with the capacity for efficient inhibition of growth, migration, and inva- sion of cancer cells would be a hopeful candidate for Figure 5 ANT2 shRNA down-regulates MMP2 and MMP9 expression and activity in SK-BR3 cells. (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 MMP2, MMP9, or GAPDH (internal control). (B) Cells were transfected with scramble shRNA or ANT2 shRNA and cultured in conditioned medium for 24 hours. Conditioned medium and cell lysates were electrophoresed in a polyacrylamide gel containing 1 mg/ml of gelatin. Proteolysis was detected as a white zone in a dark blue field. Band at 92 kDa indicates the active MMP9, 72 kDa the inactive MMP2, and 62 kDa active MMP2. use in suppression of cancer metastasis and progression, thus resulting in reduced mortality [30]. Among four isoforms of human ANT, ANT2 is speci- fically expressed in undifferentiated cells or tissues cap- able of proliferating and regenerating such as lymphocytes, kidney and liver. In addition, ANT2 expression is known to be up-regulated in several hor- mone-dependent cancers including breast and ovary cancer [31]. The overexpression of ANT2 in cancer cells was found to be associated with glycolytic metabolism of cancer cells, which raised a possible role of ANT2 during carcinogenesis [32]. In fact, we and others have shown that ANT2 knockdown decreased cell viability and induced apoptotic death in breast cancer cells with