Journal of Shanghai Jiao Tong University (Medical Science) >

2021 , Vol. 41 >Issue 6: 710 - 716

DOI: https://doi.org/10.3969/j.issn.1674-8115.2021.06.002

Basic research

Suppressing effect of the Na+/Ca2+ exchanger blocker CB-DMB on the growth of human glioblastoma cells

  • Jing-jing LIU ,
  • Hui-jie HU ,
  • Zi-kai LIU ,
  • Ming-ke SONG
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  • Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China

Online published: 2021-06-29

Supported by

National Natural Science Foundation of China(81873807);Innovative Research Team of High-level Local Universities in Shanghai(SSMU-ZDCX20181201)

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Abstract

Objective

·To test the inhibitory effect of the Na+/Ca2+ exchanger (NCX) blockers on the growth of human glioblastoma cells.

Methods

·Human glioblastoma cell lines (U87, U251 and SF188) and human astrocytes were cultured in vitro. The cells were treated with NCX blockers SN-6, YM244769, SEA0400, CB-DMB and the chemotherapeutic agent temozolomide (TMZ). SN-6, YM244769 and SEA0400 were selective inhibitors for the reverse operation of NCX; while CB-DMB was NCX bidirectional blocker, but preferentially blocked the forward mode of NCX. The TMZ was used as a reference drug. Cell counting kit-8 (CCK-8) assay was used to quantify and assess the cell viability, and half maximal inhibitory concentration (IC50) of the drug was obtained. Calcium imaging was used to detect the changes of Ca2+ signal in U87 cells treated with NCX inhibitors, and Western blotting was used to detect the expression of mitogen-activated protein kinase (MAPK) signaling pathway proteins. Cellular apoptosis was evaluated by flow cytometry assay.

Results

·CCK-8 results showed that direct application of the NCX bidirectional blocker CB-DMB to glioblastoma cell lines (U87, U251 and SF188) for 48 h caused a dose-dependent growth inhibition with IC50 values of 2.06, 2.19 and 1.82 μmol/L, respectively. In contrast, NCX reverse blockers SN-6, YM244769 and SEA0400 had no significant effect on the growth activity of glioblastoma cells. CB-DMB had little effect on the growth activity of human astrocytes. Calcium imaging and Western blotting results confirmed that CB-DMB blocked the forward transport mode of NCX to elevate intracellular Ca2+, causing intracellular calcium overload and then inducing apoptosis of U87 cells and activating MAPK signaling pathway. Flow cytometry assay results showed that the rate of apoptosis induced by CB-DMB in glioblastoma cells was much faster than that induced by TMZ (P=0.002). The combination of CB-DMB and TMZ enhanced the inhibitory effect of TMZ on the growth of tumor cells.

Conclusion

·The inhibitory effect of CB-DMB on the growth of human glioblastoma cells may be related to blocking the forward transport mode of NCX. The plasma membrane NCX is a potential new target for the treatment of human glioblastoma.

Key words： glioblastoma; Na+/Ca2+ exchanger; blocker; CB-DMB; apoptosis; temozolomide (TMZ)

Cite this article

Jing-jing LIU , Hui-jie HU , Zi-kai LIU , Ming-ke SONG . Suppressing effect of the Na+/Ca2+ exchanger blocker CB-DMB on the growth of human glioblastoma cells[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2021 , 41(6) : 710 -716 . DOI: 10.3969/j.issn.1674-8115.2021.06.002

References

1 Carter JH, McNulty SN, Cimino PJ, et al. Targeted next-generation sequencing in molecular subtyping of lower-grade diffuse gliomas: application of the World Health Organization's 2016 revised criteria for central nervous system tumors[J]. J Mol Diagn, 2017, 19(2): 328-337.
2 Pessina F, Navarria P, Cozzi L, et al. Value of surgical resection in patients with newly diagnosed grade Ⅲ glioma treated in a multimodal approach: surgery, chemotherapy and radiotherapy[J]. Ann Surg Oncol, 2016, 23(9): 3040-3046.
3 Yamashiro K, Nakao K, Ohba S, et al. Human glioma cells acquire temozolomide resistance after repeated drug exposure via DNA mismatch repair dysfunction[J]. Anticancer Res, 2020, 40(3): 1315-1323.
4 Yamamura M, Amano Y, Sakagami H, et al. Calcium mobilization during nicotine-induced cell death in human glioma and glioblastoma cell lines[J]. Anticancer Res, 1998, 18(4A): 2499-2502.
5 Robil N, Petel F, Kilhoffer MC, et al. Glioblastoma and calcium signaling: analysis of calcium toolbox expression[J]. Int J Dev Biol, 2015, 59(7/8/9): 407-415.
6 Zhang Y, Cruickshanks N, Yuan F, et al. Targetable T-type calcium channels drive glioblastoma[J]. Cancer Res, 2017, 77(13): 3479-3490.
7 Song M, Chen D, Yu SP. The TRPC channel blocker SKF 96365 inhibits glioblastoma cell growth by enhancing reverse mode of the Na+/Ca2+ exchanger and increasing intracellular Ca2+[J]. Br J Pharmacol, 2014, 171(14): 3432-3447.
8 Amoroso S, de Maio M, Russo GM, et al. Pharmacological evidence that the activation of the Na+-Ca2+ exchanger protects C6 glioma cells during chemical hypoxia[J]. Br J Pharmacol, 1997, 121(2): 303-309.
9 Hu HJ, Wang SS, Wang YX, et al. Blockade of the forward Na+/Ca2+ exchanger suppresses the growth of glioblastoma cells through Ca2+-mediated cell death[J]. Br J Pharmacol, 2019, 176(15): 2691-2707.
10 Abrunhosa-Branquinho AN, Bar-Deroma R, Collette S, et al. Radiotherapy quality assurance for the RTOG 0834/EORTC 26053-22054/NCIC CTG CEC.1/CATNON intergroup trial concurrent and adjuvant temozolomide chemotherapy in newly diagnosed non-1p/19q deleted anaplastic glioma: individual case review analysis[J]. Radiother Oncol, 2018, 127(2): 292-298.
11 Watanabe Y, Kimura J. Blocking effect of bepridil on Na+/Ca2+ exchange current in Guinea pig cardiac ventricular myocytes[J]. Jpn J Pharmacol, 2001, 85(4): 370-375.
12 Cheng H, Zhang Y, Du C, et al. High potency inhibition of hERG potassium channels by the sodium-calcium exchange inhibitor KB-R7943[J]. Br J Pharmacol, 2012, 165(7): 2260-2273.
13 Secondo A, Pannaccione A, Molinaro P, et al. Molecular pharmacology of the amiloride analog 3-amino-6-chloro-5-[(4-chloro-benzyl)amino]-n-[[(2, 4-dimethylbenzyl)-amino]iminomethyl]-pyrazinecarboxamide (CB-DMB) as a pan inhibitor of the Na+-Ca2+ exchanger isoforms NCX1, NCX2, and NCX3 in stably transfected cells[J]. J Pharmacol Exp Ther, 2009, 331(1): 212-221.
14 Zhao YT, Valdivia CR, Gurrola GB, et al. Arrhythmogenesis in a catecholaminergic polymorphic ventricular tachycardia mutation that depresses ryanodine receptor function[J]. Proc Natl Acad Sci USA, 2015, 112(13): E1669-E1677.
15 Bender S, Tang YJ, Lindroth AM, et al. Reduced H3K27me3 and DNA hypomethylation are major drivers of gene expression in K27M mutant pediatric high-grade gliomas[J]. Cancer Cell, 2013, 24(5): 660-672.
16 Tveden-Nyborg P, Bergmann TK, Lykkesfeldt J. Basic & clinical pharmacology & toxicology policy for experimental and clinical studies[J]. Basic Clin Pharmacol Toxicol, 2018, 123(3): 233-235.
17 程哲, 汪潮潮, 吴娟, 等. BFAR在胶质瘤中的表达及其与胶质瘤预后的关系[J]. 医学信息, 2021, 34(7): 78-81.
18 Li JH, Li SY, Shen MX, et al. Anti-tumor effects of Solanum nigrum L. extraction on C6 high-grade glioma[J]. J Ethnopharmacol, 2021, 274: 114034.
19 Li LL, Sun LN, Zhou HY, et al. Selective alteration of expression of Na+/Ca2+ exchanger isoforms after transient focal cerebral ischemia in rats[J]. Neurosci Lett, 2006, 404(3): 249-253.
20 Tomimoto H, Yanagihara T. Immunoelectron microscopic study of tubulin and microtubule-associated proteins after transient cerebral ischemia in gerbils[J]. Acta Neuropathol, 1992, 84(4): 394-399.
21 Zhang B, Liu B, Roos CM, et al. TRPC6 and TRPC4 heteromultimerization mediates store depletion-activated NCX1 reversal in proliferative vascular smooth muscle cells[J]. Channels (Austin), 2018, 12(1): 119-125.
22 Zhang J. New insights into the contribution of arterial NCX to the regulation of myogenic tone and blood pressure[J]. Adv Exp Med Biol, 2013, 961: 329-343.
23 Pignataro G, Gala R, Cuomo O, et al. Two sodium/calcium exchanger gene products, NCX1 and NCX3, play a major role in the development of permanent focal cerebral ischemia[J]. Stroke, 2004, 35(11): 2566-2570.
24 Hu HJ, Song M. Disrupted ionic homeostasis in ischemic stroke and new therapeutic targets[J]. J Stroke Cerebrovasc Dis, 2017, 26(12): 2706-2719.
25 Goto Y, Ogata M, Kita S, et al. YM-244769, a novel Na+/Ca2+ exchange inhibitor, efficiently improves ischemia/reperfusion-induced renal injury[J]. Biophys J, 2012, 102(3): 662a-663a.
26 Watanabe Y, Koide Y, Kimura J. Topics on the Na+/Ca2+ exchanger: pharmacological characterization of Na+/Ca2+ exchanger inhibitors[J]. J Pharmacol Sci, 2006, 102(1): 7-16.
27 White CD, Sacks DB. Regulation of MAP kinase signaling by calcium[J]. Methods Mol Biol, 2010, 661: 151-165.
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