title: Repurposing of phentolamine as a potential anticancer agent against human castration-resistant prostate cancer: A central role on microtubule stabilization and mitochondrial apoptosis pathway.
abstract: BACKGROUND
  Drug repurposing of phentolamine, an α-adrenoceptor antagonist, as an anticancer agent has been studied in human castration-resistant prostate cancer (CRPC).
  
  
  METHODS
  Cell proliferation was examined by sulforhodamine B and CFSE staining assays. Cell cycle progression and mitochondrial membrane potential (ΔΨm) were detected by flow cytometric analysis. Protein expression was detected by Western blotting. Effect on tubulin/microtubule was determined using confocal immunofluorescence microscopic examination, microtubule assembly detection, tubulin turbidity assay, and binding assay. Several assessments were used to characterize apoptotic signaling pathways and combinatory effect.
  
  
  RESULTS
  Phentolamine induced anti-proliferative effect in PC-3 and DU-145, two CRPC cell lines, and P-glycoprotein (P-gp) overexpressing cells. This effect was not significantly reduced in paclitaxel-resistant cells. Rhodamine 123 efflux assay showed that phentolamine was not a P-gp substrate. Phentolamine induced mitotic arrest of the cell cycle and formation of hyperdiploid cells, followed by an increase of apoptosis. Mitotic arrest was confirmed by cyclin B1 up-regulation, Cdk1 activation, and a dramatic increase of mitotic protein phosphorylation. Both in vitro and cellular identification demonstrated that phentolamine, similar to paclitaxel, induced tubulin polymerization and formation of multiple nuclei. Besides, it did not compete with paclitaxel binding on tubulin. Phentolamine induced the phosphorylation and degradation of Bcl-2 and Bcl-xL, two anti-apoptotic Bcl-2 family members, and the loss of ΔΨm indicating the induction of mitochondrial damage. It ultimately induced the activation of caspase-9, -8, and -3 and apoptotic cell death. Moreover, combination treatment with phentolamine and paclitaxel caused a synergistic apoptosis.
  
  
  CONCLUSIONS
  The data suggest that phentolamine is a potential anticancer agent. In contrast to a wide variety of microtubule disrupting agents, phentolamine induces microtubule assembly, leading to mitotic arrest of the cell cycle which "in turn" induces subsequent mitochondrial damage and activation of related apoptotic signaling pathways in CRPC cells. Furthermore, combination between phentolamine and paclitaxel induces a synergistic apoptotic cell death. Phentolamine has a simple chemical structure and is not a P-gp substrate. Optimization of phentolamine structure may also be a potential approach for further development.
authors.affiliation: Department of Urology, Taipei Medical University-Shuang Ho Hospital, Taipei, Taiwan.|Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Chen-Hsun
authors.initials: CH
authors.lastname: Ho
authors.affiliation: School of Pharmacy, National Taiwan University, Taipei, Taiwan.
authors.forename: Jui-Ling
authors.initials: JL
authors.lastname: Hsu
authors.affiliation: Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.|Department of Urology, National Taiwan University Hospital, Taipei, Taiwan.
authors.forename: Shih-Ping
authors.initials: SP
authors.lastname: Liu
authors.affiliation: School of Pharmacy, National Taiwan University, Taipei, Taiwan.
authors.forename: Lih-Ching
authors.initials: LC
authors.lastname: Hsu
authors.affiliation: School of Pharmacy, National Taiwan University, Taipei, Taiwan.|Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio.
authors.forename: Wei-Ling
authors.initials: WL
authors.lastname: Chang
authors.affiliation: Department of Biochemistry and Molecular Biology, Chang Gung University, Taoyuan, Taiwan.
authors.forename: Chuck C-K
authors.initials: CC
authors.lastname: Chao
authors.affiliation: School of Pharmacy, National Taiwan University, Taipei, Taiwan.
authors.forename: Jih-Hwa
authors.initials: JH
authors.lastname: Guh
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D058492:Drug Repositioning; D006801:Humans; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008870:Microtubules / Q000187:drug effects* / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D010646:Phentolamine / Q000494:pharmacology* / Q000627:therapeutic use; D064129:Prostatic Neoplasms, Castration-Resistant / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology
chemical_list: D000970:Antineoplastic Agents; D010646:Phentolamine
keywords: P-glycoprotein; castration-resistant prostate cancer; drug repurposing; microtubule stabilization; phentolamine