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.
Store-operated Ca2+ entry (SOCE) is the primary pathway of Ca2+ entry into mammalian cells. Re-modelling of the SOCE pathway has been suggested as the driving mechanism for many tumour phenotypes, such as cancer cell proliferation, migration, and metastasis. Although SOCE has been studied in many cancer types, calcium signalling, especially the SOCE pathway, is largely unexplored in medulloblastoma (MB). MB is the most common malignant paediatric brain tumour, and previously, we reported that some key SOCE components are upregulated in MB. The present study aimed to functionally characterise SOCE in MB cells. Using RT-PCR, the expression of different SOCE-regulating genes was examined cells of different MB subgroups. Our data indicate that specific subgroups of MB cells differentially express SOCE genes. For example, one key regulatory gene, ORAI1, showed a higher expression in the invasive MB subgroups 3. This difference was also reflected by a higher SOCE in these cells compared to cells from MB subgroups associated with lower invasive potential. Overall, the results highlight that distinct MB subgroups rely on differential gene expression that affects their SOCE activity. Future studies will require a functional characterisation to delineate if altered SOCE is causal for the invasiveness of MB, which will be a critical to understand the potential of SOCE as a therapeutic target for the treatment of MB.
Medulloblastoma (MB) is the most common malignant paediatric brain tumour. In our previous studies, in a screen of a panel of calcium channel modulators in our novel high-throughput 3D assay, we 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 this study, we characterized the effect of mibefradil and NNC on MB cells and elucidated 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. We also identified that CACNA1H (encoding the CaV3.2 protein) is significantly upregulated in aggressive Groups 3 and 4 MB patient samples, and its higher expression is associated with higher metastasis and lower patient survival rates. 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.
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