Sildenafil

Cervical cancer cells (HeLa, HT-3, C33A, SiHa and U14 cells) and human cervical epithelial cells (HCerEpiC) were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco's modification of Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/l penicillin, and 100 mg/l streptomycin and were cultured in a 5% CO₂ cell incubator at 37 ^oC. Sildenafil (Sigma-Aldrich; Merck KGaA, St. Louis, MO, USA) was diluted with dimethyl sulfoxide and added to the DMEM to obtain desired concentrations (0.5, 1.0, 1.5 and 2.0 μM).

The embryonal RMS cell line RD (ATCC, Manassas, VA, USA), the alveolar RMS cell line RH30 (DSMZ, Braunschweig, Germany), and the human skeletal muscle cells (SkMC; Sigma Aldrich, Taufkirchen, Germany) were routinely cultured in DMEM medium (Biochrom) supplemented with 10% FCS, 1% penicillin/streptomycin, and 1% L-glutamine (all supplements from Biochrom) in a humidified atmosphere containing 5% CO₂ at 37 °C. Cells were obtained directly from cell bank (ATCC, DSMZ, date of the procurement: 08/2020) that performs cell line characterizations and passaged in our laboratory for fewer than 6 months after receipt. All cells were tested to be mycoplasma negative (Lonza). Cells were treated with the PDE5 inhibitor Sildenafil (Sigma Aldrich; Merck KGaA, diluted in DMSO in 10 mM) in the absence and/or presence of doxorubicin for the indicated periods and with the indicated concentrations.

Ectopic homografts of 4T1 murine breast cancer cells were established in female BALB/c mice. For TOL, mice were injected with digoxin (7 mg/kg), then stimulated with PMF (80 mT at 25 Hz) for 30 min on days 1, 3 and 5 and compared with controls. Groups of TOL-treated or control mice received either losartan (20 mg/kg/d; Sigma-Aldrich) beginning 2 days before TOL or sildenafil (1 mg/kg; Sigma-Aldrich) 15 min prior to each digoxin dosing and were compared to groups of mice that did not receive supplementary drug. Tumor growth was measured every other day. Animals were euthanized when they met NIH criteria for humane endpoint euthanasia.

Icariin derivative ZW1 was synthesized as previously described^{31 (link)} and dissolved in DMSO at a concentration of 50 mM. Testosterone, letrozol, sildenafil, FSK, and protease inhibitor cocktail were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and dissolved in DMSO at a concentration of 100 mM. All solutions were stored at −20 °C. Antibodies to aromatase (CYP19A1) and GAPDH were purchased from Epitomics (Burlingame, CA, USA) and Proteintech (Rosemont, IL, USA), respectively. Rabbit monoclonal antibodies to Phospho-CREB (Ser133) and CREB (48H2) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Enzyme immunoassay (EIA): human pulmonary artery vascular smooth muscle (PAVSM) cells and growth media were obtained from Lonza Inc. (Walkersville, MD., USA). Confluent cells were pre-treated with phenol-red free DMEM (supplemented with 10% fetal calf serum) in the presence of absence of 10 mM N-acetyl-L-cysteine (NAC), a thiol reducing agent and antioxidant, to reverse mildly oxidized critical sulfhydryl groups in sGC [14 (link)] and, possibly, to prevent the oxidation of NO to NO_x; and/or 100 nM sildenafil, a PDE5 inhibitor, for 90 min followed by 500 μM H₂O₂ for 30 min. Cells were next treated with either phosphate-buffered saline or 100 μM S-nitroso-N-acetylpenicillamine (SNAP), a NO^.-donor, for 10 min. PAVSM cells were rinsed in ice-cold phosphate buffered saline and then solubilized in ice-cold 6% trichloroacetic acid. Samples were stored at -80° until the day of the assay. Samples were processed and cGMP and protein were measured as previously described [14 (link)]. cGMP formation was measured by immunoassay according to the cGMP Assay (Cayman Chemical Co., Ann Arbor, MI). H₂O₂, trichloroacetic acid, NAC, and sildenafil were purchased from Sigma-Aldrich (St. Louis, MO). Phenol-red-free DMEM was obtained from Gibco, Life Technologies, Grand Island, NY and fetal calf serum was from Atlanta biologicals, Flowery Branch, GA.