Andro

PCa cells PC3, DU145, JCA-1, TsuPr1, LNCaP and human renal tubular epithelial cells 293T and human prostate stromal cells PS30 were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). All PCa cell lines were maintained in Roswell Park Memorial Institute-1640 medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA), 100 U ml⁻¹ penicillin and 100 U ml⁻¹ streptomycin (Hyclone, Logan, UT, USA). The culture condition of 293T cells was Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA), containing 10% FBS and 1% penicillin-streptomycin (Hyclone, Logan, UT, USA). All cells were cultured at 37°C in 5% CO₂. Andro (Sigma, Milwaukee, WI, USA) was dissolved with dimethyl sulfoxide (DMSO; Sigma, Milwaukee, WI, USA), the stock solution at 100 μmol l⁻¹, and the human TRAIL (Sigma, Milwaukee, WI, USA) solution with a concentration of 100 mg ml⁻¹ was prepared by PBS (Hyclone, Logan, UT, USA) containing 0.1% bovine serum albumin (Hyclone, Logan, UT, USA). Caspases inhibitors were formulated as solutions with suitable working concentration according to the manufacturer's instructions.

T-ALL Jurkat cells were purchased from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, 10⁵ U/L penicillin, and 100 mg/L streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C in an atmosphere containing 5% CO₂. The cells were dispensed in 96-well, flat bottom microtiter plates (NUNC, Roskilde, Denmark) at a density of 1.0×10⁴ cells per well. After 24 hours (h) incubation, the cells were treated with different concentrations of Andro (0–32 μg/mL) for the indicated time periods. Then, a 20 μL aliquot of MTT solution (5.0 mg/mL) was added to each well, followed by 4 h incubation, and then an enzyme-linked immunosorbent assay (ELISA) reader (Thermo Fisher Scientific, Waltham, MA USA) was used to measure the optical density. Andro was purchased from Sigma-Aldrich Co., (St Louis, MO, USA), with a purity of ≥95% high performance liquid chromatography (HPLC). The study was approved by the ethics committee of Jining No 1 People’s Hospital.

Human breast cancer cell lines T47D (cultured in RPMI-1640) and MDA-MB-231 (cultured in Leibovitz’s L-15 medium) were obtained from ATCC. The medium contains 10% fetal bovine serum (Hyclone Laboratories Inc, UT, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). The cell lines were maintained at 37°C in a humidified incubator containing 21% O₂ and 5% CO₂. For the hypoxia experiment, cells were treated with Andro first and then incubated under 1% O₂, 94% N₂, and 5% CO₂. Andro (purity 98%) was purchased from Sigma-Aldrich Chemical Co. (St Louis, MO, USA).

Andro was obtained from Sigma–Aldrich (St Louis, MO, USA). Hydroxychloroquine (HCQ) was procured from Aladdin (Shanghai, China). Water-soluble Andrographolide sulfonate (Xi-Yan-Ping Injection) was provided by Jiangxi Qingfeng Pharmaceutical Co., Ltd (Jiangxi, China). GM6001 was purchased from MedChem Express (Monmouth Junction, NJ, USA). The IL-1β, IL-6 and TNF-α antibody were obtained from ABclonal (Wuhan, China). The primary antibodies against tumor necrosis factor-alpha receptor 2 (TNFR2), calcitonin gene-related peptide (CGRP), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4, and ADAMTS5 were from Abcam (Cambridge, UK). The CCL2 antibody was obtained from Proteintech (Chicago, IL, USA). The PE-Phalloidin and goat anti-rabbit horseradish peroxidase (HRP)-labeled secondary antibody was obtained from Fude Biological Technology (Hangzhou, China). The antibodies against p-p65, p65, and GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA).

Andro, fluoxetine, K252a (a pharmacological inhibitor of TrkB), and 5-Bromo-2-deoxyUridine (BrdU) were purchased from Sigma (St. Louis, MO). Lentiviral (LV)-TrkB-short hairpin RNA (shRNA)-enhanced green fluorescent protein (EGFP) and LV-Control-shRNA-EGFP were provided by GeneChem Co., Ltd (Shanghai, China). The doses of Andro (10, 20, 50, and 100 mg/kg), fluoxetine (20 mg/kg), and K252a (25 μg/kg) were selected based on previous reports (Chen et al., 2014 (link); Serrano et al., 2014 (link); Jiang et al., 2017 (link); Ren et al., 2017 (link); Wang et al., 2017 (link); Ni et al., 2018 (link)). Andro, fluoxetine, and K252a were dissolved in 0.9% sodium chloride (NaCl) containing 10% dimethyl sulfoxide and 20% Cremaphor EL (Vehicle). BrdU was dissolved in 0.9% NaCl. All compounds were i.p. injected at a concentration of 10 mL/kg. LV-TrkB-shRNA-EGFP and LV-Control-shRNA-EGFP were stereotaxically injected into the hippocampus.