Agelastatin A: a novel inhibitor of osteopontin-mediated adhesion, invasion, and colony formation.

Effective inhibitors of osteopontin (OPN)-mediated neoplastic transformation and metastasis are still lacking. (-)-Agelastatin A is a naturally occurring oroidin alkaloid with powerful antitumor effects that, in many cases, are superior to cisplatin in vitro. In this regard, past comparative assaying of the two agents against a range of human tumor cell lines has revealed that typically (-)-agelastatin A is 1.5 to 16 times more potent than cisplatin at inhibiting cell growth, its effects being most pronounced against human bladder, skin, colon, and breast carcinomas. In this study, we have investigated the effects of (-)-agelastatin A on OPN-mediated malignant transformation using mammary epithelial cell lines. Treatment with (-)-agelastatin A inhibited OPN protein expression and enhanced expression of the cellular OPN inhibitor, Tcf-4. (-)-Agelastatin A treatment also reduced beta-catenin protein expression and reduced anchorage-independent growth, adhesion, and invasion in R37 OPN pBK-CMV and C9 cell lines. Similar effects were observed in MDA-MB-231 and MDA-MB-435s human breast cancer cell lines exposed to (-)-agelastatin A. Suppression of Tcf-4 by RNA interference (short interfering RNA) induced malignant/invasive transformation in parental benign Rama 37 cells; significantly, these events were reversed by treatment with (-)-agelastatin A. Our study reveals, for the very first time, that (-)-agelastatin A down-regulates beta-catenin expression while simultaneously up-regulating Tcf-4 and that these combined effects cause repression of OPN and inhibition of OPN-mediated malignant cell invasion, adhesion, and colony formation in vitro. We have also shown that (-)-agelastatin A inhibits cancer cell proliferation by causing cells to accumulate in the G(2) phase of cell cycle.

Modified electroporation protocol for Lactobacilli isolated from the chicken crop facilitates transformation and the use of a genetic tool.

Isolates of Lactobacillus spp. from a collection of potentially probiotic strains isolated from the crops of broiler chickens were found to be non-electrotransformable using published techniques. One strain of Lactobacillus salivarius was shown to develop electrocompetence when an overnight culture was incubated in fresh medium. The effect was enhanced if glycine was incorporated into the fresh growth medium. When these modifications were applied to a number of other crop isolates of Lactobacillus spp., electrocompetence could be detected in approximately half the strains tested. Two temperature sensitive plasmid vectors that had been used for the genetic modification of other lactic acid bacteria were introduced into a crop strain of Lb. salivarius. Both showed temperature sensitivity at 42 degrees C and above but were relatively stable at 37 degrees C. The genetic tool harbouring an IS element allowed the delivery of the plasmid to multiple independent sites in the host chromosome. Harnessing such genetic tools will facilitate the future genetic analysis of the host bacterium.