Capsaicin (CAP) and STO-609 were purchased to Sigma-Aldrich (St. Louis, MO, USA). psPAX2 vector was a gift from Didier Trono (Addgene plasmid #12260; http://n2t.net/addgene:12260; RRID:Addgene_12260), pCMV-VSV-G was a gift from Robert Weinberg 44 (Addgene plasmid #8454; http://n2t.net/addgene:8454; RRID:Addgene_8454), pLKO.1-TRC cloning vector was a gift from from David Root 45 (Addgene plasmid #10878; http://n2t.net/addgene:10878; RRID:Addgene_10878), pMDLg/pRRE was a gift from Didier Trono 46 (Addgene plasmid #12251; http://n2t.net/addgene:12251; RRID:Addgene_12251), pRSV-Rev was a gift from Didier Trono 46 (Addgene plasmid #12253; http://n2t.net/addgene:12253; RRID:Addgene_12253) and LentiV_Neo_LKB1 was a gift from Christopher Vakoc 47 (Addgene plasmid #108111; http://n2t.net/addgene:108111; RRID: Addgene_108111).
PC3, DU-145 and LNCaP human prostate cancer cell lines were obtained from American Type Culture Collection, (ATCC CRL-1435, ATCC HTB-81 and ATCC CRL-1740 respectively) (Rockville, MD, USA). Cells were routinely grown in RPMI 1640 medium supplemented with 100 IU/ml penicillin G sodium, 100 mg/ml streptomycin sulfate, 0.25 mg/ml amphotericin B (Invitrogen, Paisley, UK) and 10% fetal bovine serum. All cell lines were incubated at 37°C in 5% CO2 and routinely tested for Mycoplasma infection. For treatment experiments, cells were plated and grown 24h, the medium was then replaced with serum-free RPMI 1640 and then incubated with different treatments for the indicated times. Cells were used at passages 4-20.
Cell proliferation/viability was determined by the MTT assay (Bio‐Rad, Richmond, CA, USA). The assay was performed in 12-well plates, according to the manufacturer's instructions (5×103/well). The absorbance was measured at 490 and 650 nm using an iMark™ Absorbance Reader from Bio-Rad (Richmond, CA, USA).
Flow cytometry for cell cycle and apoptosis.
Flow cytometry was used to detect the distribution of cell cycle. After being cultivated with the treatment, 1.5·105 cells in 35 mm culture dish were harvested in 0.35% trypsin, collected and washed with cold PBS. After that, cells were centrifuged at 500 g for 5 min and fixed with 70% cold ethanol at -20ºC overnight. Then, cells were centrifuged again and incubated in 0.5 ml PBS containing 0.1 mg/ml RNase for 30 min at 37 °C. DNA staining was performed by adding 10 μg/ml PI (Invitrogen, Eugene, Oregon, USA). Apoptosis was evaluated at 24h following treatment using an Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit BD Biosciences, San Diego, CA USA) according to the manufacturer’s instructions. Data acquisition and analysis were performed in a MACSQuant® Analyzer flow cytometry system (Miltenyi Biotec, Bergisch Gladbach, Germany) using MACSQuantify software (Miltenyi Biotec, Bergisch Gladbach, Germany). A total of 10 × 103 events were collected for each sample.
Proteins for Western blotting were isolated by lysing cells in lysis buffer [50 mM Tris pH 7.4, 0.8 M NaCl, 5 mM MgCl2, 0.1% Triton X-100] containing protease inhibitor and phosphatase inhibitor cocktail (Roche, Diagnostics; Mannheim, Germany), incubated on ice for 15 min and cleared by microcentrifugation. 20 micrograms of total protein/lane were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a PVDF membrane. Membranes were incubated overnight at 4°C with primary antibodies. After washing in T-TBS, membranes were incubated with peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (1:5000) for 2 h at room temperature. The immune complex was visualized with an ECL system (Cell Signaling Technology). Protein expression levels were quantified using Image J (National Institutes of Health, Bethesda, Maryland, USA) expressed as fold changes relative to the control treatment. Primary antibodies anti-p62, p-AMPKα1-thr172, p-ACC-ser79, pLKB1-ser428, pmTOR-ser2448 and the antibodies against the corresponding total forms were obtained from Cell Signaling Technology (Danvers, MA, USA). TRPV1 was obtained from (Thermo Scientific, MA, USA) and LC3B was from Novus (England, UK). Peroxidase labeled secondary anti-mouse IgG was from Sigma-Aldrich (St. Louis, MO, USA) and anti-rabbit IgG was from Cell Signaling Technology (Danvers, MA, USA).
Cells were transfected in 1 mL OptiMEM containing 4 μg Lipofectamine iMax (Invitrogen, Carlsbad, CA) with 100 nM LKB1‐specific small interfering RNA (siRNA) duplexes (5′-GUACUUCUGUCAGCUGAUUdTdT-3′ and 5′-AAUCAGCUGACAGAAGUACdTdT-3′) (Sigma-Aldrich, St. Louis, MO, USA) or scrambled RNA (control) according to the manufacturer's protocols (Invitrogen). At 72 h after transfection, the medium was removed and replaced with RPMI. At the indicated time points after transfection, cells were used for MTT cell viability assays or western blot analysis.
The lentiviral transduction system was used to generate cell lines with TRPV1 silencing or LKB1 overexpression. Lentivirus was produced in HEK293T cells by transfecting plasmids of interest with helper plasmids. To generate the viruses to silence TRPV1, the following mixture was added to a 10 cm dish of HEK293T at 70% confluence: 5 μg of psPAX2, 3 μg of pCMV-VSV-G, 10 μg of pLKO.1-TRC cloning vector or pLKO.1-TRC cloning vector with shTRPV1 (shTRPV1 sequence was designed from the clone ID: TRCN0000044190, Sigma, St. Louis, MO, USA) and polythylenimine (PEI) 1mg/ml at a 3:1 ratio with the total concentration of the DNA in the mix. On the other hand, to generate the viruses to overexpress LKB1, the mixture was the following: 5 μg of pMDLg/pRRE, 3 μg of pCMV-VSV-G, 2.5 μg of pRSV-Rev, 10 μg of plasmid LentiV_Neo_LKB1 and PEI 1mg/ml in the same relationship discussed above. At 6 hours after transfection, the medium was changed to fresh medium and after 48h and 72h after transfection, the supernatant with the viruses was collected, filtered through a 0.45 µm pore-size filter and used to infect PC3 and DU-145 cells, adding polybrene (1 µg/ml) (Sigma, St. Louis, MO, USA) to increase the efficiency of the infection. After infection, the cells were amplified to a larger culture surface and 24 h later they were selected with 3 µg/mL puromycin (STEMCELL Technologies) in the case of TRPV1 silencing or with 900 µg/ml G418 (Sigma, St. Louis, MO, USA) in the case of LKB1 overexpression.
The statistical analysis of the results was performed with Graph Pad Prism 9 software (San Diego, CA) using a two-way ANOVA and Tukey’s multiple comparisons test or Sidak’s multiple comparisons test. The results were reported as mean ± SD as indicated in figure caption, of at least three independent experiments. Data were considered significant when p ≤ 0.05.