Remdesivir br terminal HSP inhibitor and
terminal HSP90 inhibitor and demonstrated that it possesses strong anti-tumor activity in non-small cell lung cancer (NSCLC) via HIF-1α degradation mediated by the ubiquitin-proteasome pathway . In the present study, we sought to investigate the mechanism of action of L80 responsible for its novel effects against cell proliferation and BCSC properties in vitro, as well as angiogenesis, tumor growth and metastasis in vivo.
2. Materials and methods
2.1. Reagents and antibodies
A detailed description of the synthesis of L80 is described in our previous report . Z-VAD-fmk, Triton X-100, propidium iodide (PI), corn oil and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO). Phosphatase inhibitor and protease inhibitor cocktail tablets were purchased from Roche Applied Sciences (Penz-berg, GER). The Remdesivir that were used include: STAT3, Ki-67, CD31, ALDH1A1 (Abcam, MA); anti-AKT, phospho-AKT (Ser473), ERK, phospho-ERK (Thr202/Tyr204), JAK2, phospho-JAK2 (Tyr1007/1008), phospho-STAT3 (Tyr705), CD49f, vimentin, Nanog, Oct4 (Cell Sig-naling, CA); survivin, MEK, phospho-MEK (Ser218/222), HSP70, cyclin D1 (Santa Cruz Biotechnology, Santa Cruz,CA); β-actin (Sigma-Aldrich, Saint Louis, MO). The secondary antibodies were horseradish perox-idase (HRP)-conjugated anti-rabbit and mouse IgG (Bio-Rad Labora-tories, CA); and Alexa Fluor-594 goat anti-mouse IgG (Invitrogen, CA). r> 2.2. Breast cancer cell culture
The TNBC cell lines MDA-MB-231 (PerkinElmer, Inc. CT), Hs578T (American Type Culture Collection, ATCC), BT-549 and 4T1-Luc (Japanese Collection of Research Bioresources Cell Bank, JCRB), and the normal murine mammary gland epithelial cell line NMuMG (American Type Culture Collection) and the normal human embryonic kidney cell line HEK293 (JCRB) were cultured in MEM or RPMI 1640 (Gibco, MD) containing 10% fetal bovine serum (FBS), streptomycin-penicillin (100 U/ml) and Fungizone (0.625 μg/mL). Normal human mammary epithelial MCF10A (ATCC) cells were cultured in Mammary Epithelial Cell Growth Medium (MEGM), including hEGF, insulin, hy-drocortisone and bovine pituitary extract (SingleQuots™ Kit, Lonza, CA) containing streptomycin-penicillin (100 U/ml). Cells were incubated at 37 °C in an atmosphere of 5% CO2.
2.3. Cell viability assay
Cell viability was measured using the CellTiter 96* Aqueous One Solution Cell Proliferation Assay [MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (Promega, WI), as previously described .
Cells were stained using a FITC-conjugated Annexin V apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ) according to the manufacturer's protocol. Stained cells were analyzed by flow cytometry using a Beckman Coulter Expo (Brea, CA).
Caspase-3 activity was measured using caspase 3 colorimetric assay kits, according to the manufacturer's instructions (Sigma-Aldrich, MO). Caspase-3 activity was analyzed by absorbance at 405 nm with a Spectramax Plus384 microplate analyzer (Molecular Devices, CA).
CD44high/CD24low staining was used to identify BCSC-like cells. Cells were incubated for 30 min at 4 °C with FITC- and PE-conjugated anti-mouse IgG or FITC-conjugated anti-CD24 and PE-conjugated anti-CD44 antibodies (BD Biosciences) and analyzed by flow cytometry.
2.7. Aldefluor-positivity assay
An Aldefluor assay kit (Stemcell Technologies, Vancouver, BC) was used to assess ALDH1 activity, as previously described . As a spe-cific inhibitor of ALDH1, 50 mM diethylamino-benzaldehyde (DEAB) was used to define the Aldefluor-positive population with a flow cyt-ometer.
2.8. Western blot analysis
2.9. Allograft in vivo experiments and bioluminescence imaging
All animal procedures were carried out in accordance with animal care guidelines approved by the Korea University Institutional Animal Care and Use Committee (IACUC). Five-week-old female BALB/c mice were obtained from the Shizuoka Laboratory Animal Center (Shizuoka, Japan) and housed in a specific pathogen-free environment. The ani-mals were acclimated for 1 week prior to the study and had free access to food and water. 1 × 105 cells from 4T1 mammospheres were im-planted subcutaneously in the right flank of 6-week-old BALB/c female mice (n = 8/each group). After 1 week, vehicle (DMSO/corn oil, 1:9) or L80 (20 mg/kg/day, every other day) was administered in-traperitoneally for 27 days, and tumor volumes were measured using a caliper and calculated using formula V=(Length × Width2)/2. The animals were then anesthetized and subjected to NightOWL LB983 bioluminescence imaging (BLI) (Berthold Technologies, TN). The pro-cedures were performed as previously described .