Inhibition of p110α or of the downstream PI3K signaling pathway components PDK1 and Akt, as well as phosphoinositide sequestration, blocks invadopodia formation in breast cancer cells.

Delivery ofcholesterol to inner mitochondrial membranes is rate-limiting for steroidogenesis in the zona fasciculata of adrenal cortex. A protein that stimulates this process was isolated to homogeneity from bovine adrenal tissue. This protein's primary structure has been determined in its entirety by a combination of automated Edman microsequencing, fast-atom bombardment mass spectrometry (FAB-MS). The sequence was identical to that previously reported for bovine brain endozepine, except that it lacks the last two residues, -Gly-Ile, at the C terminus. To our knowledge, isolation of an endozepine-related protein from a tissue other than brain has not been reported previously. Endozepine competes with benzodiazepines for saturable binding sites in synaptosomes and in mitochondria of specific peripheral tissues. Previous reports have localized the adrenal benzodiazepine receptor to the outer mitochondrial membrane. In this report, we show that the prototypic benzoiazepine, diazepam, effects a stimulation of adrenal mitochondrial cholesterol delivery similar to that observed for endozepine. The effective diazepam concentration was consistent with that previously shown to displace a high-affinity ligand of the mitochondrial benzodiazepine receptor. The action of diazepam in adrenal mitochondria suggests that the mediation of corticotropininduced steroidogenesis may be the physiological function of the peripheral-type benzodiazepine receptor. These studies provide new insights into the previously unknown function of peripheral benzodiazepine receptors and should allow new investigations into the stimulation of steroidogenesis by endozepines and benzodiazepines in the brain and in certain peripheral tissues.

Novel π-conjugated polymers with a bithiazole unit and various acceptor units were synthesized by polycondensation via Pd-catalyzed direct arylation. The results of polycondensation reactions depend on chalcogen elements in the monomers. The presence of a Se atom inhibited polymerization, presumably owing to the coordination of the Se moiety to the Pd center; the issue was resolved by increasing the reaction temperature (120 °C). The chalcogen elements also affected the interchain interactions of the obtained polymers. The strong interchain interactions led to low solubility, high crystallinity, and a large red-shift of absorption in the film state compared with that in the solution state. Absorption spectra and DFT calculations revealed that the bithiazole units served as weak donor units in case that the bithiazole units were directly connected to strong acceptor units. The combination of the bithiazole units with the strong acceptor units afforded deep HOMO−LUMO levels.

Invadopodia are ventral cell protrusions formed in invasive cancer cells. Because invadopodia have extracellular matrix (ECM) degradation activity, they are thought to function in cancer invasion. In this study, we examined the roles of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] and PI(4,5)P 2 -producing enzymes in invadopodia formation in MDA-MB-231 human breast cancer cells. Immunofluorescence analysis showed that PI(4,5)P 2 accumulates at invadopodia on the ventral cell surface. Injection of an anti-PI(4,5)P 2 antibody inhibited invadopodia formation along with gelatin degradation activity. Sequestering of PI(4,5)P 2 by overexpression of the phospholipase C (PLC) d1-pleckstrin homology (PH) domain, a specific probe for PI(4,5)P 2 , also blocked invadopodia formation, while a mutated PLCd1-PH domain that lacks PI(4,5)P 2 -binding activity had no effect. Cellular PI(4,5)P 2 production is mainly mediated by type-I phosphatidylinositol 4-phosphate 5-kinase (PIP5KI) family proteins, which include PIP5KIa, Ib, and Ic. Real-time quantitative PCR analysis showed that PIP5KIa is a dominant isoform expressed in MDA-MB-231 cells. Knockdown of PIP5KIa by small-interfering RNA (siRNA) inhibited invadopodia formation and gelatin degradation. Immunofluorescence analysis revealed that endogenous PIP5KIa protein localizes at invadopodia, which is corroborated by the observation that exogenously expressed green fluorescent protein (GFP)-fused PIP5KIa protein also accumulates at gelatin degradation sites. These results indicate that localized production of PI(4,5)P 2 by PIP5KIa is required for invadopodia formation and ECM degradation by human breast cancer cells. (Cancer Sci 2010; 101: 1632-1638 I nvadopodia are extracellular matrix (ECM)-degrading protrusions formed at the ventral surface of invasive cancer cells.(1,2) In the case of breast cancer cell lines, the ability to form invadopodia is closely related to their invasive and metastatic properties.(3,4) Additionally, during intravasation, invadopodia-like protrusions in tumor cells have been observed in vivo by intravital imaging.(5,6) Therefore, invadopodia are proposed to function in local ECM degradation during cancer invasion and metastasis, although the in vivo relevance of invadopodia has yet to be determined. To date, many components of invadopodia have been reported, including proteins involved in the regulation of the actin cytoskeleton, cell signaling, cell-ECM adhesion, ECM degradation, and membrane remodeling.(7-9) We and other researchers previously proposed that invadopodia formation occurs in several steps. Invadopodia precursors are assembled by an actin polymerization machinery containing neural Wiskott-Aldrich syndrome protein (N-WASP), the Arp2 ⁄ 3 complex, and cortactin in response to extracellular stimuli. (3,10) The invadopodia precursors are then stabilized by further actin polymerization induced by cofilin and Mena, and finally these structures gather matrix metalloproteinases to mature into functional invadopodia. (3,11,12) However, it i...

We have purified to homogeneity a protein from bovine adrenal (fasciculata) cells that is capable of: 1) stimulating synthesis of pregnenolone (in a concentration-dependent fashion) by mitochondria when added with exogenous cholesterol; 2) increasing the concentration of cholesterol in outer and inner membrane when added to mitochondria with exogenous cholesterol; 3) increasing the rate of transport of cholesterol from outer membrane to inner membrane when the two membranes are incubated together in an aqueous buffer and subsequently separated by centrifugation; and 4) increasing the proportion of molecules of C27 side-chain cleavage P-450 that are bound to the substrate cholesterol. The protein is homogeneous according to two-dimensional polyacrylamide gels, shows a molecular weight of 8200 and is therefore referred to as 8.2 K. It is proposed that 8.2 K may be involved in the regulation of steroid synthesis in bovine fasciculata cells, possibly by stimulating entry of cholesterol into the outer membrane and by altering the intramitochondrial distribution of this substrate.