Abstract
Glioblastoma (GB) is associated with poor patient survival owing to uncontrolled tumor proliferation and resistance to apoptosis. Human ether-a-go-go-related gene K+ channels (hERG; Kv11.1, KCNH2) are expressed in multiple cancer cells including GB and control cell proliferation and death. We hypothesized that pharmacological targeting of hERG protein would inhibit tumor growth by inducing apoptosis of GB cells. The small molecule hERG ligand doxazosin induced concentration-dependent apoptosis of human LNT-229 (EC50 = 35 µM) and U87MG (EC50 = 29 µM) GB cells, accompanied by cell cycle arrest in the G0/G1 phase. Apoptosis was associated with 64% reduction of hERG protein. HERG suppression via siRNA-mediated knock down mimicked pro-apoptotic effects of doxazosin. Antagonism of doxazosin binding by the non-apoptotic hERG ligand terazosin resulted in rescue of protein expression and in increased survival of GB cells. At the molecular level doxazosin-dependent apoptosis was characterized by activation of pro-apoptotic factors (phospho-erythropoietin-producing human hepatocellular carcinoma receptor tyrosine kinase A2, phospho-p38 mitogen-activated protein kinase, growth arrest and DNA damage inducible gene 153, cleaved caspases 9, 7, and 3), and by inactivation of anti-apoptotic poly-ADP-ribose-polymerase, respectively. In summary, this work identifies doxazosin as small molecule compound that promotes apoptosis and exerts anti-proliferative effects in human GB cells. Suppression of hERG protein is a crucial molecular event in GB cell apoptosis. Doxazosin and future derivatives are proposed as novel options for more effective GB treatment.
Highlights
Glioblastoma (GB) is the most common malignant primary brain tumor in adults
Apoptosis of LNT-229 cells was analyzed in situ by transferase-mediated dUTP nick end labeling (TUNEL) fluorescence, assessing DNA damage and fragmentation as characteristic apoptotic features
Propidium iodide co-staining revealed that the fraction of late apoptotic cells yielded 11.964.7% (20 mM; n = 4; p = 0.033; Figure 2B) and 20.663.9% (40 mM; n = 3; p = 0.025; Figure 2C) after treatment with doxazosin compared to 3.161.2% (n = 4) under control conditions (Figure 2A)
Summary
Glioblastoma (GB) is the most common malignant primary brain tumor in adults. Current treatment is based on maximal safe surgical resection, followed by chemo- and radiotherapy when feasible [1]. Outcome is poor despite optimal therapy with a mean survival rate of 1 year following diagnosis, which is due to uncontrolled tumor proliferation, infiltrative growth, angiogenesis, and resistance to apoptosis and medical treatment [2,3]. HERG channels control cell proliferation and apoptosis [12]. Targeting of hERG channels by the small molecule a1adrenoceptor antagonist doxazosin induces apoptosis in vitro independent of its anti-adrenergic function [13,14,15]. This proapoptotic mechanism of action was extended to structurally unrelated compounds, suggesting broader significance [11,16]. In addition to the heart, hERG K+ channels are expressed in multiple types of cancer cells including GB (reviewed in [12])
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