Cyclic Imine Pinnatoxin G is Cytotoxic to Cancer Cell Lines via Nicotinic Acetylcholine Receptor-Driven Classical Apoptosis.

Pinnatoxin G is a cyclic imine neurotoxin produced by dinoflagellates that has been reported in shellfish. Like other members of the pinnatoxin family, it has been shown to have its effects via antagonism of the nicotinic acetylcholine receptors, with preferential binding to the α7 subunit often upregulated in cancer. Because increased activity of α7 nicotinic acetylcholine receptors contributes to increased growth and resistance to apoptosis, the effect of pinnatoxin G on cancer cell viability was tested. In a panel of six cancer cell lines, all cell types lost viability, but HT29 colon cancer and LN18 and U373 glioma cell lines were more sensitive than MDA-MB-231 breast cancer cells, PC3 prostate cancer cells, and U87 glioma cells, correlating with expression levels of α7, α4, and α9 nicotinic acetylcholine receptors. Some loss of cell viability could be attributed to cell cycle arrest, but significant levels of classical apoptosis were found, characterized by caspase activity, phosphatidylserine exposure, mitochondrial membrane permeability, and fragmented DNA. Intracellular Ca2+ levels also dropped immediately upon pinnatoxin G treatment, which may relate to antagonism of nicotinic acetylcholine receptor-mediated Ca2+ inflow. In conclusion, pinnatoxin G can decrease cancer cell viability, with both cytostatic and cytotoxic effects.

Mesenchymal stem cell homing towards cancer cells is increased by enzyme activity of cathepsin D.

Mesenchymal stem cells home towards inflammatory microenvironments, such as the tumour stroma, where they have been shown to have both pro- and anti-tumorigenic effects. Here, we demonstrate that the aspartic acid protease cathepsin D is part of the chemoattraction process. Using a Boyden chamber co-culture system, the migration of the mesenchymal stem cells and their invasion through Matrigel increased in the presence of breast cancer MDA-MB-231 cells, colon cancer HT29 cells or their conditioned media. Mesenchymal stem cell movement was reduced by protease inhibitors of matrix metalloproteinases and by pepstatin A, an inhibitor of cathepsin D. We confirmed a role for cathepsin D through addition of recombinant protein, upregulation of cathepsin D release using chloroquine and knockdown of cathepsin D expression. While all cell types expressed active cathepsin D, enzymatically inactive precursor procathepsin D was expressed only at low levels by mesenchymal stem cells. Expression in mesenchymal stem cells was increased following co-culture with cancer cells. The chemoattractive effect of cathepsin D required its enzymatic activity, but not changes in mesenchymal stem cell proliferation or adhesion rates. In conclusion, cathepsin D and its precursors enhance mesenchymal stem cell homing towards tumour sites, most likely by enzymatic mechanisms.

Mesenchymal stem cells inhibit breast cancer cell migration and invasion through secretion of tissue inhibitor of metalloproteinase-1 and -2.

Mesenchymal stem cells (MSCs) form part of tumor stroma, and are typically considered to be pro-tumorigenic, promoting continuing tumor growth and metastasis. Here, we describe a mechanism by which MSCs may be anti-tumorigenic, through inhibition of breast cancer cell migration and invasion, an important part of metastasis. MDA-MB-231 and T47D cells were co-cultured in a Transwell insert above MSCs or MSC conditioned media and their migration or invasion through Matrigel measured. The conditioned media was found to inhibit breast cancer cell movement. TIMP-1 and TIMP-2, inhibitors of matrix metalloproteinases (MMPs), were identified as candidates for this inhibition and enhanced secretion of MMPs was not sufficient to counter the anti-migratory effects of TIMP expression. Inhibition of TIMP activity showed that TIMP-1 and TIMP-2 together were largely responsible for the reduction of migration and invasion by MSCs. Therefore, MSCs may play anti-tumorigenic, anti-metastatic roles in tumor development, with the overall outcome dependent upon the balance of pro-and anti-tumorigenic molecules secreted.

The type 2 anti-Müllerian hormone receptor has splice variants that are dominant-negative inhibitors.

Anti-Müllerian hormone (AMH) has both paracrine and hormonal actions that occur at different AMH concentrations, and in cells with different densities of its specific receptor (Amhr2). This diversity is not explained by canonical AMH signaling. We report that Amhr2 has two splice variants: Amhr2Δ2 (AMH binding site) and Amhr2Δ9/10 (kinase domain). Both spliced variants inhibit AMH signaling in a reporter assay. The mRNA for the spliced variants was relatively less abundant than Amhr2 mRNA in all tissues. This suggests that the physiological function(s) of the receptor variants may be restricted to specific cellular/subcellular sites and/or to the transport of AMH.