Verapamil augments carmustine- and irradiation-induced senescence in glioma cells by reducing intracellular reactive oxygen species and calcium ion levels

Abstract

Resistance to conventional therapies and frequent recurrence are the major obstacles to the treatment of high-grade gliomas, including glioblastoma. Thus, the development of new therapeutic strategies to overcome these obstacles is necessary to improve the treatment outcomes. In this study, we found that verapamil, a pan–adenosine triphosphate–binding cassette transporter and L-type voltage-dependent calcium channel inhibitor, sensitized U87MG glioma cells to carmustine- and irradiation-induced senescence. Furthermore, our results indicated that verapamil treatment, in combination with carmustine and irradiation, rendered U87MG glioma cells and several patient-derived glioma stem cells more sensitive to therapy-induced senescence than individual or dual-combination treatments. When investigating the underlying mechanism, we found that verapamil treatment markedly decreased intracellular reactive oxygen species and calcium ion levels. Reactive oxygen species reduction with N-acetylcysteine, a reactive oxygen species scavenger, rendered U87MG glioma cells more sensitive to carmustine and irradiation whereas the protein kinase C agonist, phorbol 12-myristate 13-acetate, mitigated the effects of carmustine and irradiation. Taken together, our results indicate that verapamil may be a potent therapeutic sensitizer for increasing the effectiveness of glioblastoma treatment.

Introduction

Glioblastoma is one of the most aggressive brain tumors, which accounts for more than 60% of malignant glioma cases. ¹ Glioblastomas exhibit multiple features of tumor malignancy; they are extremely proliferative and histologically highly vascularized by active angiogenesis, necrotic and infiltrate to normal brain tissue. ¹ Many oncogenes, such as epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor (PDGFR) downstream signaling nodes, are abnormally activated by amplification or point mutation of the genes. And the typical loss-of-function mutations of tumor suppresser genes include deletion or point mutation of TP53, INK4A and PTEN.^1,2

Currently, standard treatment protocols of glioblastoma are surgical resection followed by adjuvant radiotherapy and chemotherapy with temozolomide.^3,4 However, complete surgical resection of tumor bulk is often impeded by high invasive capability of glioblastoma. Furthermore, despite the recent development of anti-cancer therapeutic drugs such as chemical inhibitors or monoclonal antibodies specifically targeting oncogenic signaling pathways, only a few have been applied to the glioblastoma patients. ⁵ The cells consisting glioblastoma bulk are known to be heterogeneous; each of the cells carry diverse genetic mutations, ⁶ thereby giving rise to a population which is resistant to not only conventional but also molecular target therapies. Besides, delivery of the drugs to the brain could be blocked by blood–brain barrier surrounding brain blood vessels, thereby limiting the choice of the therapeutic modalities. ⁷ Accordingly, the majority of glioblastoma patients suffer disease recurrence after standard treatment protocols and thereby present extremely short 12–15 months median survival after diagnosis. ¹ Therefore, alternative therapeutic regimens are needed to improve therapeutic response for glioblastoma patients.

It is generally accepted that glioblastomas contain a small population of glioma stem cells (GSCs) that possess several characteristics commonly attributed to normal stem cells, including self-renewal and differentiation. Moreover, these GSCs are known to be the major cause of tumor initiation, progression, and relapse after conventional treatment. ⁸ Notably, GSCs have been proven to be more resistant to chemotherapy than their differentiated progeny owing to their increased expression of multifunctional efflux adenosine triphosphate (ATP)-binding cassette (ABC) transporters.^9,10 In addition, because of their preferential activation of the DNA damage repair system, GSCs are less sensitive to radiation treatment. ¹¹ Although a number of studies have identified mechanisms underlying therapeutic resistance in glioblastomas, there is an urgent need to develop novel therapeutic strategies to increase treatment success.

Reactive oxygen species (ROS) are generated within cells through several intrinsic mechanisms, such as the respiratory chain, as well as extrinsic stimuli, such as ionizing radiation. ¹² In general, tumor cells have higher levels of intracellular ROS than normal cells. ¹² This observation leads to a hypothetical therapeutic option of targeting tumor cells by selectively increasing ROS levels. Unfortunately, normal cells are more sensitive to ROS, leading to stress-induced cell death due to severe damage to the DNA, protein, and membranes. Therefore, given that tumor cells require higher levels of ROS for their proliferation, it is more likely that eliminating, rather than increasing, ROS in tumor cells would be an effective adjuvant strategy for targeting tumor cells in combination with conventional anti-cancer therapies. ¹³

Intracellular calcium ion concentrations are tightly controlled by intrinsic mechanisms, including release of endoplasmic reticulum (ER)-sequestered calcium, influx from outside cells, and efflux out of cells. ¹⁴ Elevated intracellular calcium concentrations affect multiple downstream signals, including mitogen-activated protein kinases (MAPKs), calmodulin, and protein kinase C (PKC), which in turn promote cell proliferation and inhibit cell death.^14,15

Verapamil, a first-generation drug for nervous system disorders and coronary artery diseases, blocks L-type voltage-dependent calcium channels to reduce intracellular calcium concentrations. In addition, it is known to act as a pan-ABC transporter inhibitor, blocking the efflux of various anti-cancer drugs, thereby enhancing the efficacy of the drugs. ¹⁶

In this study, because of the bi-directional inhibitory capability of verapamil, we evaluated verapamil as an adjuvant agent for the treatment of glioblastoma in combination with the anti-cancer drug carmustine and irradiation. We found that verapamil increased the carmustine- and irradiation-induced senescence of glioma cells and GSCs by decreasing intracellular ROS and calcium ion levels.

Materials and methods

Cell line and cell culture

The U87MG glioma cell line was purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Lonza, Switzerland) supplemented with fetal bovine serum (10%; Biotechnics Research, Lake Forest, CA, USA), penicillin/streptomycin (1%; Life Technologies, Carlsbad, CA, USA), l-glutamine (2 mmol/L; Life Technologies), and gentamicin (50 µg/mL; Cellgro, Manassas, VA, USA). Patient-derived GSCs were kindly provided by Dr Ichiro Nakano. ¹⁷ GSCs were cultured in DMEM/F12 (Lonza) supplemented with B27 (0.04%; Invitrogen, Waltham, MA, USA), epidermal growth factor (EGF; 20 ng/mL; R&D Systems, Minneapolis, MN, USA), basic fibroblast growth factor (bFGF; 20 ng/mL; R&D Systems), penicillin/streptomycin (1%), l-glutamine (2 mmol/L), and gentamicin (50 µg/mL).

Gamma irradiation and chemical treatment

Cells were harvested from culture plates and stained with trypan blue solution (Invitrogen) to count the living cells. Living cells were then irradiated with defined doses of ¹³⁷Cs gamma rays (3 Gy in Figures 2–4 and 7, 5 Gy in Figure 6.) using an IBL-473C (Pharmalucence, Billerica, MA, USA). Verapamil, N-acetylcysteine (NAC), carmustine (also known as bis-chloroethylnitrosourea, (BCNU)), and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell death and senescence analysis

Cell death was measured by staining with AnnexinV-FITC (BD Biosciences, East Rutherford, NJ, USA) and propidium iodide (BD Biosciences) followed by flow cytometry analysis using FACSVerse (BD Biosciences). Cellular senescence was measured using a senescence-associated β-galactosidase (SA-β-gal) staining kit (Cell Signaling Technology, Danvers, MA, USA). Microscopic images of SA-β-gal-positive cells were obtained with an inverted microscope (Carl Zeiss, Germany) and the positively stained cells were counted. Pseudo-colored images of SA-β-gal-positive cells were also generated using MetaMorph Offline Software (Molecular Devices, Sunnyvale, CA, USA).

Intracellular ROS detection

Cells were harvested from the culture plate, washed with phosphate-buffered saline (PBS, Sigma-Aldrich), and incubated in 5 mM dichlorofluorescein (DCF, Invitrogen) at 37°C for 5 min. Flow cytometry to examine DCF-positive cells was performed using FACSVerse (BD Biosciences).

Intracellular calcium detection

Intracellular calcium levels were measured using a Fluo-4 NW Calcium Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and stained with Alizarin Red S (Sigma-Aldrich). Cells were treated with irradiation in combination with defined doses of verapamil or NAC in 96-multiwell plates (SPL Life Sciences, Korea). At each time points, the cells were incubated in Fluo-4 NW Calcium Assay solution at 37°C, then the fluorescence emission per well was detected and total cell count per well was quantified with an IncuCyte ZOOM system (Essen BioScience, Ann Arbor, MI, USA). Fluorescence intensity per well was divided by total cell count per well to normalize the decreased number of the cells by treatment of irradiation, verapamil, and NAC. For Alizarin Red S staining, cells were treated with irradiation in combination with verapamil or NAC. After 3 days, the cells were washed with PBS, fixed in the 10% neutral-buffered formalin solution (Sigma-Aldrich) for 30 min, and washed with distilled water. The cells were then incubated with 2% Alizarin Red S solution (pH 4.2) in the room temperature for 45 min, washed with distilled water, and then the microscopic image was obtained with an inverted microscope (Carl Zeiss). Alizarin Red S intensity per field was measured using MetaMorph Offline Software (Molecular Devices) and was divided by cell count per field.

Statistical analysis

Data were analyzed with two-tailed Student’s t tests. Results with p values less than 0.05 were considered statistically significant. Data are presented as the mean ± standard deviation.

Results

Verapamil increases carmustine-induced glioma cell senescence

We examined whether verapamil would display a synergistic effect on glioma cell death after treating with conventional chemotherapy and ionizing gamma irradiation (IR). Since temozolomide exerts a cytostatic rather than a cytotoxic effect, we used carmustine, a cytotoxic alkylating agent, to examine chemotherapeutic-induced outcomes. ¹⁸ We first treated U87MG glioma cells with carmustine alone or with both carmustine and verapamil. The results indicated that combined treatment with carmustine and verapamil increased cell death compared to carmustine treatment alone (Figure 1(a)). However, as cell death rates were lower than expected at less than 20% with the combined treatment, we also examined premature senescence, a common feature of cancer cells that underwent cytotoxic damage. ¹⁹ We evaluated whether cellular senescence was increased by carmustine treatment and further increased by combined treatment with carmustine and verapamil. After treatment with carmustine alone, 23% of U87MG glioma cells displayed senescent cell features, measured by SA-β-gal activity, ²⁰ whereas 43% of U87MG glioma cells treated with both carmustine and verapamil were SA-β-gal-positive (Figure 1(b) and (d)). Furthermore, 62% of U87MG glioma cells expressed a different senescence cell marker, nuclear p21^CIP1 cyclin-dependent kinase inhibitor, ²¹ after combined treatment with carmustine and verapamil (Figure 1(c) and (d)), compared to less than 40% of those treated with carmustine alone. Although verapamil alone did not induce notable cell death and senescence in U87MG glioma cells (Figure 1(a)–(d)), we further examined whether verapamil exerts cytotoxic effect on non-tumor cells. Immortalized human astrocytes were treated with verapamil of same dose as treated previously; however, they exhibited neither senescence nor cell death upon verapamil treatment (Figure 1(e) and (f)). These results indicate that verapamil primarily sensitized U87MG glioma cells to carmustine-induced cellular senescence rather than directly increasing cell death.

Figure 1.

Verapamil increases U87MG glioma cell senescence after treatment with the anti-cancer drug carmustine. (a) Cell death was measured in the absence or presence of verapamil following treatment with or without 1 µg/mL carmustine. (b) Quantification of SA-β-gal-positive U87MG glioma cells in the absence or presence of verapamil following treatment with or without 1 µg/mL carmustine. (c) Quantification of immunofluorescence staining against p21^CIP1 in U87MG glioma cells in the absence or presence of verapamil following treatment with or without 1 µg/mL carmustine. (d) Representative images of (b) and (c). SA-β-gal staining images are presented as bright-field microscopic images (top) and pseudo-colored images (bottom). (e) Representative images of SA-β-gal staining in human astrocyte in the absence or presence of 10 µM verapamil. (f) Cell death was measured in the absence or presence of 10 µM verapamil. Microscopic images are 200× magnified (*p < 0.05; **p < 0.01; ***p < 0.001 (n = 3)). Data are represented as the mean ± standard deviation.

Verapamil increases irradiation-induced glioma cell senescence

Similarly, we measured U87MG glioma cell death after IR treatment and found that neither IR treatment alone nor combined treatment with verapamil caused significant changes in cell death (Figure 2(a)). However, 23% of U87MG glioma cells displayed SA-β-gal activity after treatment with IR alone, whereas 54% of U87MG glioma cells were SA-β-gal-positive after treatment with both IR and verapamil (Figure 2(b) and (d)). In addition, 56% of U87MG glioma cells expressed nuclear p21^CIP1 after treatment with both IR and verapamil compared to 27% after treatment with IR alone (Figure 2(c) and (d)). These results indicate that verapamil also sensitized U87MG glioma cells to radiation-induced cellular senescence.

Figure 2.

Verapamil induces U87MG glioma cell senescence after treatment with IR. (a) Cell death was measured in the absence or presence of verapamil following treatment with or without 3 Gy IR. (b) Quantification of SA-β-gal-positive U87MG glioma cells in the absence or presence of verapamil following treatment with or without 3 Gy IR. (c) Quantification of immunofluorescence staining against p21^CIP1 in U87MG glioma cells in the absence or presence of verapamil following treatment with or without 3 Gy IR. (d) Representative images of (b) and (c). SA-β-gal staining images are presented as bright-field microscopic images (top) and pseudo-colored images (bottom). Microscopic images are 200× magnified (**p < 0.01; ***p < 0.001; N.S. indicates statistically not significant (n = 3)). Data are represented as the mean ± standard deviation.

Verapamil-mediated decrease in intracellular ROS sensitizes glioma cells to radiation-induced senescence

To identify underlying mechanisms of verapamil-mediated sensitization to anti-cancer therapy–induced glioma cell senescence, we investigated how verapamil sensitizes U87MG glioma cells to IR-induced cellular senescence. We chose to look at IR treatment because verapamil increased IR-induced senescence more than carmustine-induced senescence. A previous study indicated that radiation sensitivity is regulated by intracellular ROS levels; cancer cells with low levels of intracellular ROS are more resistant to IR than those with high levels of intracellular ROS. ²² We evaluated the intracellular ROS levels of U87MG glioma cells after IR treatment in the presence or absence of verapamil. DCF staining revealed that intracellular ROS levels decreased in U87MG glioma cells 2 days after verapamil treatment (Figure 3(a) and (b)). In addition, intracellular ROS levels decreased to a greater extent in irradiated U87MG glioma cells upon concurrent treatment of verapamil (Figure 3(a) and (b)). To verify whether IR-induced senescence in glioma cells can be attributed to decreased ROS levels, we treated U87MG glioma cells with NAC, an ROS scavenger, and found that NAC treatment effectively reduced intracellular ROS levels in U87MG glioma cells (Figure 3(c) and (d)). Moreover, combined treatment with IR and NAC led to significant increases in cellular senescence relative to IR treatment alone (Figure 3(c)). Taken together, these results suggest that verapamil sensitized glioma cells to radiation-induced senescence by decreasing intracellular ROS levels.

Figure 3.

Verapamil and NAC augment glioma cell senescence by reducing ROS production. (a) DCF-flow cytometry assay was used to detect intracellular ROS levels in U87MG glioma cells treated with 3 Gy IR in the presence or absence of 10 µM verapamil. *p < 0.05 indicates significant differences as compared to the control group of each time points; #p < 0.05 indicates significant differences as compared to the IR group of each time points (n = 3). (b) Representative histogram plot showing the result of DCF-flow cytometry in (a). Arrow indicates the shift of peak fluorescence intensity in the irradiated U87MG glioma cells. (c) DCF fluorescence intensities showing intracellular ROS levels in the U87MG glioma cells following treatment with 3 mM NAC. *p < 0.05 (n = 3). (d) Representative histogram plot showing the result of DCF-flow cytometry in (c). (e) Quantification of SA-β-gal-positive U87MG glioma cells following treatment with or without 3 Gy IR in the presence of NAC. ***p < 0.001 (n = 3). Bar graph data represent the mean ± standard deviation.

Verapamil decreases intracellular calcium concentration resulting in increased carmustine- and IR-induced senescence

Since verapamil acts as an L-type calcium channel inhibitor and the concentration of calcium ions is closely related to the survival and growth of the cell, we investigated whether differences in intracellular calcium ion levels render glioma cells sensitive to carmustine- and IR-induced cellular senescence. We evaluated the levels of intracellular calcium ions after verapamil treatment, with or without IR. The results indicated that verapamil effectively decreased intracellular calcium ion levels in U87MG glioma cells independent of IR treatment (Figure 4(a)). Interestingly, although NAC treatment alone did not significantly decrease intracellular calcium ion levels as measured by fluorometric detection method, when combined with IR, calcium ion levels dramatically reduced compared to both control and irradiated condition (Figure 4(b)). In addition, we evaluated the intracellular calcium ion levels by colorimetric detection method using Alizarin Red S and observed similar reduction of calcium ion levels upon treatment with either verapamil or NAC, independent of IR treatment (Figure 4(c) and (d)). These results suggest that intracellular calcium ion levels might be associated with glioma cell senescence induced by concurrent treatment of verapamil and IR or carmustine.

Figure 4.

Verapamil and NAC abate calcium production in U87MG glioma cells. Intracellular calcium ion level was measured following treatment with (a) 3 Gy IR in the presence or absence of 10 µM verapamil and (b) 3 Gy IR in the presence or absence of 3 mM NAC. *p < 0.05 indicates significant differences as compared to the control group of each time points; #p < 0.05 indicates significant differences as compared to the IR group of each time points (n = 3). (c) U87MG glioma cells were stained with Alizarin Red S 3 days after irradiation in combination with verapamil or NAC. (d) Quantification of Alizarin Red S staining intensity in (c). *p < 0.05; **p < 0.01 (n = 3). Data represent the mean ± standard deviation.

Activation of PKC alleviates chemotherapy- and radiotherapy-induced glioma cell senescence

Intracellular calcium ions activate PKC, which in turn activates multiple pathways related to cell proliferation and survival. ¹⁴ Given that both verapamil and NAC promoted senescence and decreased intracellular calcium ion levels in U87MG glioma cells, we examined whether PKC activation would reduce carmustine- and IR-induced cellular senescence. We treated U87MG glioma cells with PMA, a known PKC activator, along with carmustine. SA-β-gal assay showed that combined treatment with PMA and carmustine resulted in significantly less SA-β-gal-positive U87MG glioma cells compared to carmustine treatment alone (Figure 5(a) and (c)). In addition, nuclear p21^CIP1-positive U87MG glioma cells markedly decreased after combined treatment with PMA and carmustine (Figure 5(b) and (c)).

Figure 5.

PMA reduces carmustine-induced senescence in U87MG glioma cells. (a) Quantification of SA-β-gal-positive U87MG glioma cells in the absence or presence of PMA following treatment with or without 3 µg/mL carmustine. (b) Quantification of immunofluorescence staining against p21^CIP1 in U87MG glioma cells in the absence or presence of PMA following treatment with or without 3 µg/mL carmustine. (c) Representative images of (a) and (b). SA-β-gal staining images are presented as bright-field microscopic images (top) and pseudo-colored images (bottom). Microscopic images are 200× magnified (***p < 0.001 (n = 3)). Data are represented as the mean ± standard deviation.

Similarly, we treated U87MG glioma cells with PMA in combination with IR and evaluated cellular senescence. Concurrent treatment of PMA and IR resulted in significantly decreased SA-β-gal-positive cells (Figure 6(a) and (c)) and nuclear p21^CIP1-positive cells (Figure 6(b) and (c)) compared to IR alone. Taken together, these results indicate that verapamil sensitized glioma cells to carmustine- and IR-induced senescence by decreasing intracellular calcium ion levels.

Figure 6.

PMA reduces IR-induced senescence in U87MG glioma cells. (a) Quantification of SA-β-gal-positive U87MG glioma cells in the absence or presence of PMA following treatment with or without 5 Gy IR. (b) Quantification of immunofluorescence staining against p21^CIP1 in U87MG glioma cells in the absence or presence of PMA following treatment with or without 5 Gy IR. (c) Representative images of (a) and (b). SA-β-gal staining images are presented as bright-field microscopic images (top) and pseudo-colored images (bottom). Microscopic images are 200× magnified. ***p < 0.001 (n = 3). Data represent the mean ± standard deviation.

Verapamil sensitizes glioma cells and GSCs to concurrent chemotherapy- and radiotherapy-induced senescence

A number of clinical studies have shown that concurrent treatment with chemotherapy and radiotherapy is more effective for patients with glioblastoma than chemotherapy or radiotherapy alone.^4,23 Therefore, we examined whether verapamil could sensitize glioma cells and GSCs to the combination therapy. U87MG glioma cells and four glioblastoma patient-derived GSCs (84NS GSCs, 19 GSCs, 157 GSCs, and 528NS GSCs) were treated with carmustine, IR, and verapamil in single, double, or triple combinations. Cellular senescence was then measured by SA-β-gal staining. Notably, although single and double treatments increased SA-β-gal-positive cells, triple combination treatment with carmustine, IR, and verapamil showed the most significant increase in SA-β-gal-positive cells in both U87MG glioma cells (Figure 7(a)) and all four GSCs (Figure 7(b)–(e)). Taken together, these results indicate that verapamil rendered glioma cells and primary GSCs sensitive to therapy-induced cellular senescence, and that inhibitors of ABC transporters and L-type calcium channels might be potent therapeutic sensitizers for patients with glioblastoma.

Figure 7.

Verapamil accelerates senescence of glioma cells and glioma stem cells by combination treatment with carmustine and IR. Quantification of SA-β-gal-positive cells was determined in (a) U87MG glioma cells and the patient-derived GSCs, (b) 84NS GSCs, (c) 19 GSCs, (d) 157 GSCs, and (e) 528NS GSCs, following single, double, or triple combination treatment with carmustine (1 µg/mL in (a) and 0.5 µg/mL in (b–e)), IR (3 Gy), and verapamil (5 µM). All the p values defined by Student’s t test between triple combination and the others were less than 0.05 (marked by *), except for carmustine plus IR versus triple combination in Figure 7(b) (marked by #). Data represent the mean ± standard deviation.

Discussion

Targeting anti-cancer therapy resistance is a significant priority for prolonged survival of patients with malignant gliomas because therapy resistance is considered as the major cause of tumor relapse following conventional therapies; however, a clinically relevant targeting modality for therapy-resistant gliomas is not yet available. Here, we suggest a novel therapeutic strategy that sensitizes glioma cells and GSCs to chemotherapy and radiotherapy. Through in vitro experiments, we demonstrated that verapamil promotes IR-induced and anti-cancer drug–induced senescence in glioma cells and GSCs by reducing intracellular ROS and calcium ion levels.

Verapamil treatment led to decrease in intracellular ROS level in glioma cells independent of IR treatment. Mitochondria are major cellular organelles that regulate basal intracellular ROS levels by balancing generation and scavenging activity. Many ABC transporters exist on mitochondrial membranes. Thus, the inactivation of mitochondrial ABC transporters with verapamil, a pan-ABC transporter inhibitor, may lead to aberrant endogenous ROS levels that could eventually render glioma cells more sensitive to radiation and chemotherapy. ²⁴

Also, it is known that the calcium ion concentration is closely related to ROS homeostasis. Calcium ions promote ROS production by regulating the activity of multiple modulators, including the mitochondrial respiratory chain and membrane nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. ²⁵ Inversely, ROS is known to increase intracellular calcium ion levels by activating calcium release channels in the ER, ²⁶ which is in line with our observations that treatment of calcium channel inhibitor verapamil resulted in reduction of intracellular ROS (Figure 3(a) and (b)), and NAC, a precursor of the antioxidant glutathione, decreased intracellular calcium ion levels (Figure 4(b) and (d)). Thus, we speculate that the verapamil induces calcium ion depletion, which in turn reduces intracellular ROS. Furthermore, decreased intracellular ROS further diminishes intracellular calcium ions.

A number of studies have suggested that ROS and PKC signaling pathways promote cellular senescence.^27,28 However, ample evidence has demonstrated that ROS promotes tumor cell survival and proliferation by activating MAPK, phosphoinositide 3-kinase (PI3K)/AKT and nuclear factor kappa B (NFκB) signaling pathways. ²⁹ In addition, PKC is also known to activate these signaling pathways.^14,30,31 Therefore, it is likely that verapamil-mediated reduction of ROS and calcium ions might induce chemotherapy- and radiotherapy-induced glioma cell senescence by mitigating these signaling pathways.

Because verapamil is a first-generation drug for nervous system disorders, several side effects, including cytotoxicities, have been reported.^16,32 However, the doses used in this study were considerably lower and exhibited no direct effects on senescence or cell death in human normal astrocytes and glioma cells. Thus, it is likely that the concentrations used in this study achieve the desired response without causing detrimental side effects. In this study, we demonstrated that verapamil was synergistic with conventional therapies by simultaneously blocking ABC transporters and reducing ROS and calcium ions to sensitize glioma cells to chemotherapy and radiation. Furthermore, because verapamil is already approved for clinical use, our proposed therapeutic strategy to eliminate glioma cells using concurrent treatment with verapamil, radiation, and chemotherapy is suitable for use in malignant glioma patients. However, because our in vitro experimental results suggest possibilities about use of verapamil or other ABC transporter inhibitors as an adjuvant for conventional chemotherapy and radiotherapy, the effects of this therapeutic strategy should be validated in physiological aspects through in vivo experimental approaches.