Histone deacetylase inhibitors, such as phenylbutyrate, are currently undergoing clinical trials as potential anticancer agents. Phenylbutyrate can induce cell differentiation and apoptosis in a number of cancer cell types and can act in synergy with ionizing radiation and chemotherapy to induce apoptosis. We used the sea urchin embryo basement membrane invasion assay to show that phenylbutyrate potently inhibited the invasive properties of both prostate and breast cancer cells at clinically achievable doses. This inhibition was dose-dependent and persisted for at least 24 hr after the drug was removed. These results suggest that in addition to activating apoptosis in cancer cells, phenylbutyrate may be used in prevention of metastatic disease. 4 -7 It has been shown that butyrate and phenylbutyrate can act in synergy with radiation 8 -11 or chemotherapy 11,12 to induce cells to undergo apoptosis. Furthermore, some HDAC inhibitors have recently been found to inhibit angiogenesis. 13 The anti-angiogenic properties of HDAC inhibitors may occur through the down regulation of VEGF expression. 10 In our previous study we found that phenylbutyrate reduced the expression of caveolin-1. 10 Although the exact role of caveolin-1 in carcinogenesis is unclear, caveolin-1 is thought to regulate integrin signaling 14 and high expression of caveolin-1 is associated with a metastatic phenotype in breast and prostate cancer. [15][16][17] In addition to reducing caveolin-1 expression, phenylbutyrate has been found to reduce the expression of the invasion-related plasminogen activator, urokinase, in glioma cells 18 and reduce the invasiveness of cancer cells in artificial matrigel chamber invasion assays. 18,19 We explored the ability of phenylbutyrate to block the invasiveness of metastatic prostate and breast cancer cells using the sea urchin embryo basement membrane invasion assay. This invasion assay utilizes the natural occurring, selectively permeable basement membranes of sea urchin embryos (SU-ECM) as invasion substrates. 20,21 The sea urchin embryo basement membrane is functionally analogous to the mammalian basement membranes underlying the epidermis because it is invaded by pigment cells during development. 22 We have shown previously that these SU-ECM invasion substrates can be selectively invaded by metastatic tumor cells. 20 In fact, we have shown that this assay can predict the metastatic potential of cancer cell lines with greater than 99% confidence. 20 We show that phenylbutyrate effectively inhibits the invasiveness of metastatic prostate and breast cancer cells in the SU-ECM invasion assay.
MATERIAL AND METHODS
Cell cultures and phenylbutyrate treatmentsPC3 and DU-145 cells were obtained from American Type Culture Collection (Manassas, VA) and Steve Ethier, University of Michigan, kindly provided us with the SUM-149 cell line. The PC3 and DU-145 cells were cultured in MEM (Life Technologies, Grand Island, NY) supplemented with 10% FBS (HyClone, Logan, UT), with 1ϫ amino acids, vitamins and antibiotics. SUM-1...