Elsevier

Biochemical Pharmacology

Volume 85, Issue 7, 1 April 2013, Pages 888-897
Biochemical Pharmacology

Inhibition of T-type calcium channels disrupts Akt signaling and promotes apoptosis in glioblastoma cells

https://doi.org/10.1016/j.bcp.2012.12.017Get rights and content

Abstract

Glioblastoma multiforme (GBM) are brain tumors that are exceptionally resistant to both radio- and chemotherapy regimens and novel approaches to treatment are needed. T-type calcium channels are one type of low voltage-gated channel (LVCC) involved in embryonic cell proliferation and differentiation; however they are often over-expressed in tumors, including GBM. In this study, we found that inhibition of T-type Ca2+ channels in GBM cells significantly reduced their survival and resistance to therapy. Moreover, either T-type selective antagonists, such as mibefradil, or siRNA-mediated knockdown of the T-type channel alpha subunits not only reduced cell viability and clonogenic potential, but also induced apoptosis. In response to channel blockade or ablation, we observed reduced phosphorylation of Akt and Rictor, suggesting inhibition of the mTORC2/Akt pathway. This was followed by reduction in phosphorylation of anti-apoptotic Bad and caspases activation. The apoptotic response was specific for T-type Ca2+ channels, as inhibition of L-type Ca2+ channels did not induce similar effects. Our results implicate T-type Ca2+ channels as distinct entities for survival signaling in GBM cells and suggest that they are a novel molecular target for tumor therapy.

Introduction

Glioblastoma multiforme (GBM) are malignant tumors of the brain with an exceedingly poor prognosis (median of 10–12 months with radiation and chemotherapy, 14.6 months overall [1]). Current treatment consists of surgical excision followed by chemo- and radiotherapy. However, a majority of GBM are phosphatase and tensin homolog (PTEN)-deficient, many harbor mutations in p53, epidermal growth factor receptor (EGFR) and other genetic alterations, which confer resistance to apoptosis and therapy [2]. Consequently, there is a need for new approaches to therapy.

Calcium (Ca2+) is a crucial secondary messenger that regulates many cellular processes, among which are cell proliferation and survival [3], [4]. Voltage-dependent Ca2+ channels provide one of the pathways for influx of calcium into cells, and channel activation leads to transient elevation in the concentration of Ca2+ in the cytosol. Among these channels, the low voltage-gated Ca2+ channel family (LVCC or Cav3, commonly called T-type Ca2+ channels) are functionally linked to many physiological processes [5]. For example, T-type channels are normally expressed in the brain and regulate neuronal excitablility and sensory transmission [6]. On the other hand, T-type Ca2+ channels are often over-expressed in GBM and other cancer cells [7], [8], and are thought to support tumor proliferation and progression [9], [10]. According to Human Protein Atlas (http://www.proteinatlas.org), majority of GBM tumor samples obtained from patients expressed LVCC Cav3.1 (IHC staining: strong 9%, moderate 18%, weak 55%, negative 18%), while 27% expressed Cav3.2 (IHC staining: weak 27%, negative 73%); Cav3.3 expression was not determined. Therefore, LVCC pose an attractive potential target for GBM tumor therapy. Therefore, T-type channels pose an attractive potential target for cancer therapy, either using specific antagonists as a single agent, or in combination with standard chemo- or radiotherapy [8], [11].

Among the several known calcium channel antagonists available, the majority are either those with broad specificity or with specificity for L-type channels [12]. Only a few specific T-type channel antagonists exist. Mibefradil (Posicor/Ro 40-5967, Hoffmann-La Roche) was originally developed for treating hypertension and chronic angina pectoris [13], [14] and is a potent inhibitor of T-type channel currents with 10 to 20 times higher selectivity for T-type over L-type Ca2+ channels [15]. Mibefradil was FDA-approved and extensively used but later recalled from the market because of concerns over potential toxicity due to drug-drug interactions [16]. Nonetheless, it is well-tolerated and has high efficacy as a T-type channel antagonist. Thus, there has been recent interest in re-purposing mibefradil as an anti-cancer compound [11].

Here we examine the function of T-type Ca2+ channels in glioblastoma cells. We show that inhibition of T-type channels with mibefradil, or down-regulation of T-type channel subunits expression with siRNA, leads to an increase in apoptosis and sensitizes glioblastoma cells to ionizing radiation in vitro. We also demonstrate reduced mTORC2/Akt signaling in the cells following inhibition of T-type Ca2+ channels. Our results raise the possibility that T-type channel inhibitors may be useful as novel and effective therapeutics for glioblastomas.

Section snippets

Cell lines and reagents

Human GBM cell lines, U251, U87 and T98G, were purchased from the American Type Culture Collection (ATCC; Rockville, MD) and maintained in a 37 °C/5% CO2 humidified chamber in RPMI-1640 medium supplemented with 5% FBS (U251), or in MEM medium with 10% FBS (U87 and T98G). All cell culture materials and supplies were purchased from Life Technologies GIBCO (Grand Island, NY). No authentication of cell lines was done by the authors. Mibefradil and TTL1177 (formerly TH1177, [17]) were generous gifts

Inhibition of T-type Ca2+ channels induces apoptotic cell death in U251 GBM cells

We observed that mibefradil reduced the viability of U251 glioblastoma cells in a dose-dependent manner (Fig. 1A), with an EC50 of 3.5 ± 0.20 μmol/L. Cells treated with mibefradil failed to exclude Trypan blue, an indication of increased membrane permeability, a marker of cytostatic effect. The effect was dose- (Fig. 1B, first panel) and time-dependent (Fig. 1B, second panel). Moreover, cells treated with mibefradil were unable to recover even after re-plating into fresh, drug-free medium. Cells

Discussion

Our study shows that inhibition of T-type calcium channels in glioblastoma cells leads to apoptotic cell death. We present evidence that these effects occur in response to either pharmacological inhibition or to siRNA-mediated knockdown of T-type Ca2+ channel subunits. This response is channel type-specific, seeing as inhibition of L-type Ca2+ channels did not elicit the same effects. We found that reduced T-type channel activity leads to a reduction in phosphorylation of the mTORC2 component,

Conflict of interest

Dr. Lloyd S. Gray is a co-founder of and consultant to TAU Therapeutics, LLC.

Acknowledgments

This work was supported in part by the UVa CCSG P30 CA44579 grant, the James and Rebecca Craig Fund and the UVa Department of Radiation Oncology George P. Amorino pilot grant (to JD).

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