title: Testosterone induces apoptosis in vascular smooth muscle cells via extrinsic apoptotic pathway with mitochondria-generated reactive oxygen species involvement.
abstract: Testosterone exerts both beneficial and harmful effects on the cardiovascular system. Considering that testosterone induces reactive oxygen species (ROS) generation and ROS activate cell death signaling pathways, we tested the hypothesis that testosterone induces apoptosis in vascular smooth muscle cells (VSMCs) via mitochondria-dependent ROS generation. Potential mechanisms were addressed. Cultured VSMCs were stimulated with testosterone (10(-7) mol/l) or vehicle (2-12 h) in the presence of flutamide (10(-5) mol/l), CCCP (10(-6) mol/l), mimetic manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP; 3 × 10(-5) mol/l), Z-Ile-Glu(O-ME)-Thr-Asp(O-Me) fluoromethyl ketone (Z-IETD-FMK; 10(-5) mol/l), or vehicle. ROS were determined with lucigenin and dichlorodihydrofluorescein; apoptosis, with annexin V and calcein; O2 consumption, with a Clark-type electrode, and procaspases, caspases, cytochrome c, Bax, and Bcl-2 levels by immunoblotting. Testosterone induced ROS generation (relative light units/mg protein, 2 h; 162.6 ± 16 vs. 100) and procaspase-3 activation [arbitrary units, (AU), 6 h; 166.2 ± 19 vs. 100]. CCCP, MnTMPyP, and flutamide abolished these effects. Testosterone increased annexin-V fluorescence (AU, 197.6 ± 21.5 vs. 100) and decreased calcein fluorescence (AU, 34.4 ± 6.4 vs. 100), and O2 consumption (nmol O2/min, 18.6 ± 2.0 vs. 34.4 ± 3.9). Testosterone also reduced Bax-to-Bcl-2 ratio but not cytochrome-c release from mitochondria. Moreover, testosterone (6 h) induced cleavage of procaspase 8 (AU, 161.1 ± 13.5 vs. 100) and increased gene expression of Fas ligand (2(ΔΔCt), 3.6 ± 1.2 vs. 0.7 ± 0.5), and TNF-α (1.7 ± 0.4 vs. 0.3 ± 0.1). CCCP, MnTMPyP, and flutamide abolished these effects. These data indicate that testosterone induces apoptosis in VSMCs via the extrinsic apoptotic pathway with the involvement of androgen receptor activation and mitochondria-generated ROS.
authors.affiliation: Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Brazil; rheurealves@usp.br.
authors.forename: Rheure Alves Moreira
authors.initials: RA
authors.lastname: Lopes
authors.affiliation: Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil; and.
authors.forename: Karla Bianca
authors.initials: KB
authors.lastname: Neves
authors.affiliation: Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil; and.
authors.forename: Cezar Rangel
authors.initials: CR
authors.lastname: Pestana
authors.affiliation: Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Brazil;
authors.forename: André Lima
authors.initials: AL
authors.lastname: Queiroz
authors.affiliation: Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Brazil;
authors.forename: Camila Ziliotto
authors.initials: CZ
authors.lastname: Zanotto
authors.affiliation: Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Brazil;
authors.forename: Andréia Z
authors.initials: AZ
authors.lastname: Chignalia
authors.affiliation: Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil; and.
authors.forename: Yara Maria
authors.initials: YM
authors.lastname: Valim
authors.affiliation: Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Brazil; School of Physical Education and Sports of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil.
authors.forename: Leonardo R
authors.initials: LR
authors.lastname: Silveira
authors.affiliation: Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil; and.
authors.forename: Carlos
authors.initials: C
authors.lastname: Curti
authors.affiliation: Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Brazil;
authors.forename: Rita C
authors.initials: RC
authors.lastname: Tostes
mesh_terms: D000726:Androgen Antagonists / Q000494:pharmacology; D000728:Androgens / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000378:metabolism; D005485:Flutamide / Q000494:pharmacology; D008297:Male; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010101:Oxygen Consumption / Q000187:drug effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*; D011944:Receptors, Androgen / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013739:Testosterone / Q000494:pharmacology*; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D000726:Androgen Antagonists; D000728:Androgens; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D011944:Receptors, Androgen; D051028:bcl-2-Associated X Protein; D013739:Testosterone; D005485:Flutamide; D020169:Caspases
keywords: ROS generation; apoptosis; mitochondria; testosterone