Doxazosin, a Classic Alpha 1-Adrenoceptor Antagonist, Overcomes Osimertinib Resistance in Cancer Cells via the Upregulation of Autophagy as Drug Repurposing

Abstract

Osimertinib, which is a third-generation epidermal growth factor receptor tyrosine kinase inhibitor, is an important anticancer drug because of its high efficacy and excellent safety profile. However, resistance against osimertinib is inevitable; therefore, therapeutic strategies to overcome the resistance are needed. Doxazosin, a classic quinazoline-based alpha 1-adrenoceptor antagonist is used to treat hypertension and benign prostatic hyperplasia with a known safety profile. The anticancer effects of doxazosin have been examined in various types of malignancies from the viewpoint of drug repositioning or repurposing. However, it currently remains unclear whether doxazosin sensitizes cancer cells to osimertinib. Herein, we demonstrated that doxazosin induced autophagy and enhanced the anticancer effects of osimertinib on the cancer cells and cancer stem cells of non-small cell lung cancer, pancreatic cancer, and glioblastoma at a concentration at which the growth of non-tumor cells was not affected. The osimertinib-sensitizing effects of doxazosin were suppressed by 3-methyladenine, an inhibitor of autophagy, which suggested that the effects of doxazosin were mediated by autophagy. The present study provides evidence for the efficacy of doxazosin as a combination therapy with osimertinib to overcome resistance against osimertinib.

Keywords: autophagy; cancer stem cells; doxazosin; drug repositioning; drug repurposing; drug resistance; osimertinib.

Figure 1

Doxazosin induces cancer cell death and inhibits cancer cell growth without affecting non-tumor cells. A549, PANC-1, differentiated GS-Y01 (differentiated glioma stem cells induced by serum in culture medium), PC-9-OR (subline from PC-9, PC-9 osimertinib resistant) (a) and IMR-90 (normal human fibroblasts) cells (b) were treated with the indicated concentrations of doxazosin (DOX) for 3 days, and the numbers of viable and dead cells (left panels), the percentage of dead cells (center panels), and the growth inhibition rate (right panels) were assessed. Values in the graphs represent the means ± SD of triplicate samples of a representative experiment repeated with similar results. * p < 0.05.

Figure 2

Doxazosin sensitizes several types of cancer cells to osimertinib. Cells were treated with/without 2 µM osimertinib (OSI) with/without 15 µM doxazosin (DOX) for 3 days, and the numbers of viable and dead cells (left panels), the percentage of dead cells (center panels), and the growth inhibition rate (right panels) were assessed. Values represent the means ± SD of triplicate samples of a representative experiment repeated with similar results. * p < 0.05.

Figure 3

Doxazosin activates autophagy. Cells were treated (a) with/without 2 µM osimertinib (OSI) (A549 cells); (b) with/without 15 µM doxazosin (DOX); or (d) with/without doxazosin in the presence/absence of 10 nM bafilomycin A1 (BAF) (A549 cells), for 3 days, and then subjected to an immunoblot analysis for autophagic markers. A549 cells were treated with/without 15 µM DOX for 3 days, and then subjected to an immunofluorescence analysis (c). The relative density was shown below each band of immunoblot.

Figure 4

An autophagy inhibitor partially reverses the induction of autophagy and osimertinib-sensitizing effects of doxazosin. (a) A549 cells were treated with/without 15 µM doxazosin (DOX) and with/without 0.5 mM 3-methyladenine (3-MA) for 3 days, and then subjected to an immunoblot analysis; (b) A549 cells were treated with/without 15 µM DOX, with/without 2 µM osimertinib (OSI), and with/without 0.5 mM 3-MA for 3 days, and the numbers of viable and dead cells (left panel), the percentage of dead cells (center panel), and the growth inhibition rate (right panel) were assessed. Values represent the means ± SD of triplicate samples of a representative experiment repeated with similar results. * p < 0.05. The relative density was shown below each band of immunoblot.