T-Type Calcium Channel Inhibitors Induce Apoptosis in Medulloblastoma Cells Associated with Altered Metabolic Activity

Abstract

Medulloblastoma (MB) is the most common malignant paediatric brain tumour. In our previous studies, we developed a novel 3D assay for MB cells that was used to screen a panel of plasma membrane calcium channel modulators for their effect on the 3D growth of D341 MB cells. These studies identified T-type (CaV3) channel inhibitors, mibefradil and NNC-55–0396 (NNC) as selective inhibitors of MB cell growth. Mibefradil was originally approved for the treatment of hypertension and angina pectoris, and recently successfully completed a phase I trial for recurrent high-grade glioma. NNC is an analogue of mibefradil with multiple advantages compared to mibefradil that makes it attractive for potential future clinical trials. T-type channels have a unique low voltage-dependent activation/inactivation, and many studies suggest that they have a direct regulatory role in controlling Ca2+ signalling in non-excitable tissues, including cancers. In our previous study, we also identified overexpression of CaV3.2 gene in MB tissues compared to normal brain tissues. In this study, we aimed to characterise the effect of mibefradil and NNC on MB cells and elucidate their mechanism of action. This study demonstrates that the induction of toxicity in MB cells is selective to T-type but not to L-type Ca2+ channel inhibitors. Addition of CaV3 inhibitors to vincristine sensitised MB cells to this MB chemotherapeutic agent, suggesting an additive effect. Furthermore, CaV3 inhibitors induced cell death in MB cells via apoptosis. Supported by proteomics data and cellular assays, apoptotic cell death was associated with reduced mitochondrial membrane potential and reduced ATP levels, which suggests that both compounds alter the metabolism of MB cells. This study offers new insights into the action of mibefradil and NNC and will pave the way to test these molecules or their analogues in pre-clinical MB models alone and in combination with vincristine to assess their suitability as a potential MB therapy.

Pharmacological characterisation of store‑operated calcium entry in medulloblastoma cell lines

“…Recently, we studied the effect of T-type Ca 2+ channel inhibitors mibefradil and NNC-55-0396 on MB cells' growth and survival. Inhibition of T-type Ca 2+ channels induces apoptosis in MB cells and their altered metabolic activity [33]. Given the importance of calcium signalling in general and the rising evidence for the role of SOCE in cancer, we studied this particular pathway in MB cell lines.…”

T-Type Calcium Channels: A Mixed Blessing

“…Moreover, it decreased cell viability in PC-3 cells, in an in-vitro model of neuroendocrine prostate cancer and in an in vivo model of glioblastoma, prolonging animal survival [ 106 , 109 ]. However, later reports showed that mibefradil can inhibit other ion channels, including the voltage-gated sodium, Ca 2+ - and volume-activated chloride channels, and potassium (inward rectifier, delayed rectifier, and hERG) channels in the sinus and atrioventricular nodes, slowing conduction velocity [ 48 , 135 ].…”

“…In particular, NNC 55-0396, an analog of mibefradil with higher blood-brain-barrier permeability, is more selective, and it blocks especially Cav3.1 and Cav3.2 with lower non-specific effects on L-type Ca 2+ channels. Similar to mibefradil, NNC 55-0396 altered mitochondrial function, energy metabolism, induced cellular apoptosis in medulloblastoma cells [ 135 ], and caused cell-cycle arrest in a wide range of melanoma cells [ 136 ]. In some cases, the concentrations at which tetralols proved to be toxic for cultured cancer cells are higher than those required to block T-type Ca 2+ channels.…”