Adenylate cyclase (AC) toxin from Bordetella pertussis delivers its catalytic domain to the interior of target cells where it converts host ATP to cAMP in a process referred to as intoxication. This toxin also hemolyzes sheep erythrocytes by a mechanism presumed to include pore formation and osmotic lysis. Intoxication and hemolysis appear at strikingly different toxin concentrations and evolve over different time scales, suggesting that different molecular processes may be involved. The present study was designed to test the hypothesis that intoxication and hemolysis occur by distinct mechanisms.Although the hemolytic activity of AC toxin has a lag of >1 h, intoxication starts immediately. Because of this difference, we sought a surrogate or precursor lesion that leads to hemolysis, and potassium efflux has been observed from erythrocytes treated with other poreforming toxins. AC toxin elicits an increase in K ؉ efflux from sheep erythrocytes and Jurkat cells, a human Tcell leukemia line, that begins within minutes of toxin addition. The toxin concentration dependence along with the analysis of the time course suggest that toxin monomers are sufficient to elicit release of K ؉ and to deliver the catalytic domain to the cell interior. Hemolysis, on the other hand, is a highly cooperative event that likely requires a subsequent oligomerization of these individual units. Although induction of K ؉ efflux shares some structural and environmental requirements with both intoxication and hemolysis, it can occur under conditions in which intoxication is reduced or prevented. The data presented here suggest that the transmembrane pathway by which K ؉ is released is separate and distinct from the structure required for intoxication but may be related to, or a precursor of, that which is ultimately responsible for hemolysis.

Glioblastoma stem-like cells (GSC) promote tumor initiation, progression and therapeutic resistance. Here we show how GSC can be targeted by the FDA approved drug mibefradil which inhibits the T-type calcium channel Cav3.2. This calcium channel was highly expressed in human GBM specimens and enriched in GSC. Analyses of the TCGA and REMBRANDT databases confirmed upregulation of Cav3.2 in a subset of tumors and showed that overexpression associated with worse prognosis. Mibefradil treatment or RNAi-mediated attenuation of Cav3.2 was sufficient to inhibit the growth, survival and stemness of GSC, and also sensitized them to temozolomide (TMZ) chemotherapy. Proteomic and transcriptomic analyses revealed that Cav3.2 inhibition altered cancer signaling pathways and gene transcription. Cav3.2 inhibition suppressed GSC growth in part by inhibiting pro-survival AKT/mTOR pathways and stimulating pro-apoptotic survivin and BAX pathways. Further, Cav3.2 inhibition decreased expression of oncogenes (PDGFA, PDGFB, and TGFB1) and increased expression of tumor suppressor genes (TNFRSF14 and HSD17B14). Oral administration of mibefradil inhibited growth of GSC-derived GBM murine xenografts, prolonged host survival and sensitized tumors to TMZ treatment. Our results offer a comprehensive characterization of Cav3.2 in GBM tumors and GSC, and provide a preclinical proof of concept for repurposing mibefradil as a mechanism-based treatment strategy for GBM.

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T-type calcium channels are involved in a multitude of cellular processes, both physiological and pathological, including cancer. T-type channels are also often aberrantly expressed in different human cancers and participate in the regulation of cell cycle progression, proliferation, migration, and survival. Here, we review the recent literature and discuss the controversies, supporting the role of T-type Ca(2+) channels in cancer cells and the proposed use of channels blockers as anticancer agents. A growing number of reports show that pharmacological inhibition or RNAi-mediated downregulation of T-type channels leads to inhibition of cancer cell proliferation and increased cancer cell death. In addition to a single agent activity, experimental results demonstrate that T-type channel blockers enhance the anticancer effects of conventional radio- and chemotherapy. At present, the detailed biological mechanism(s) underlying the anticancer activity of these channel blockers is not fully understood. Recent findings and ideas summarized here identify T-type Ca(2+) channels as a molecular target for anticancer therapy and offer new directions for the design of novel therapeutic strategies employing channels blockers. Physiological relevance: T-type calcium channels are often aberrantly expressed or deregulated in cancer cells, supporting their proliferation, survival, and resistance to treatment; therefore, T-type Ca(2+) channels could be attractive molecular targets for anticancer therapy.

Bordetella pertussis adenylate cyclase (AC) toxin belongs to the RTX family of toxins but is the only member with a known catalytic domain. The principal pathophysiologic function of AC toxin appears to be rapid production of intracellular cyclic AMP (cAMP) by insertion of its catalytic domain into target cells (referred to as intoxication). Relative to other RTX toxins, AC toxin is weakly hemolytic via a process thought to involve oligomerization of toxin molecules. Monoclonal antibody (MAb) 3D1, which binds to an epitope (amino acids 373 to 399) at the distal end of the catalytic domain of AC toxin, does not affect the enzymatic activity of the toxin (conversion of ATP into cAMP in a cell-free system) but does prevent delivery of the catalytic domain to the cytosol of target erythrocytes. Under these conditions, however, the ability of AC toxin to cause hemolysis is increased three-to fourfold. To determine the mechanism by which the hemolytic potency of AC toxin is altered, we used a series of deletion mutants. A mutant toxin, ⌬AC, missing amino acids 1 to 373 of the catalytic domain, has hemolytic activity comparable to that of wild-type toxin. However, binding of MAb 3D1 to ⌬AC enhances its hemolytic activity three-to fourfold similar to the enhancement of hemolysis observed with 3D1 addition to wild-type toxin. Two additional mutants, ⌬N489 (missing amino acids 6 to 489) and ⌬N518 (missing amino acids 6 to 518), exhibit more rapid hemolysis with quicker onset than wild-type toxin does, while ⌬N549 (missing amino acids 6 to 549) has reduced hemolytic activity compared to wild-type AC toxin. These data suggest that prevention of delivery of the catalytic domain or deletion of the catalytic domain, along with additional amino acids distal to it, elicits a conformation of the toxin molecule that is more favorable for hemolysis.