br Fig Effect of L treatment on HSP
Fig. 3. Effect of L80 treatment on HSP90 client proteins in TNBC cells. (A) Immunoblot analyses of AKT, phospho-AKT (Ser473), MEK, phospho-MEK (Ser218/222), ERK, phospho-ERK (Thr202/Tyr204) and HSP70 protein expression in MDA-MB-231 and 4T1 LY294002 following exposure to L80 (1–5 μM, 72 h). Actin was used as a loading control. Quantitative graphs represent the ratio of phosphorylated-/total-proteins, and the ratio of HSP70/actin in the presence or absence of L80 (bottom panel, *p < 0.05). (B) Effect of L80 (1–5 μM, 72 h) on expression of STAT3 signaling-related factors in TNBC cells in vitro. Quantitative graphs represent the ratio of phosphorylated-/total-proteins, and the ratio of survivin/actin and cyclin D1/actin in the presence or absence of L80 (bottom panel, *p < 0.05). The results are presented as mean ± SEM of at least three independent experiments analyzed by one-way ANOVA followed by Bonferroni's post hoc test.
2.10. Immunohistochemistry and in-situ localization of apoptosis (TUNEL)
with fluorescence-conjugated secondary antibody at RT for 2 h, fol-lowed by ProLong gold antifade reagent with DAPI (Life Technologies, CA). Phospho-JAK2 (1:100) and phospho-STAT3 (1:100) staining was performed using a Vectastain ABC kit (Vector Laboratories, CA) with diaminobenzidine (DAB, GBI labs, WA) followed by hematoxylin counterstaining (Sigma, MO) in accordance with the manufacturer's instructions. In situ TUNEL was carried out on tissue sections using a
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Fig. 4. L80 suppresses BCSC-like properties. (A–B) After exposure to L80 (1–10 μM, 72 h), CD44high/CD24low populations and ALDH1 activity in MDA-MB-231 cells were evaluated by flow cytometry. The quantitative graph represents the percentage of CD44high/CD24low populations (A, *p < 0.05) and Aldefluor-positive cells (B, **p < 0.01) are shown in the right panels, respectively. (C) MDA-MB-231 (1 × 105 cells/ml) and 4T1 (5 × 104 cells/ml) were cultured in serum-free suspension conditions in ultralow attachment plates in the presence or absence of L80 (2 and 5 μM) for 4 days. The number and volume of mammospheres was quantified by optical microscopy (right panels, ***p < 0.001). (D) Effect of L80 on mammosphere formation in an allograft model with 4T1 cells in vivo. Dissociated single cells (1 × 105/ml) from allograft tumors (300–350 mm3) were plated in ultralow attachment dishes and cultured in the presence or absence of L80 (5 μM) for 4 days. The number and volume of mammospheres was quantified (right panels, ***p < 0.001). Data were analyzed by Student's t-test or one-way ANOVA followed by Bonferroni's post hoc test.
TUNEL kit (Roche Applied Sciences, Penzberg, GER) in accordance with the manufacturer's instructions.
2.11. Mammosphere formation in vitro and in vivo assay
In vitro and in vivo mammosphere-forming assays were performed as previously described . In brief, cells were plated in ultralow at-tachment dishes and cultured in HuMEC basal serum free medium (Gibco, MD), supplemented with B27 (1:50, Invitrogen), 20 ng/mL basic fibroblast growth factor (bFGF, Sigma), 20 ng/mL human epi-dermal growth factor (EGF, Sigma), 4 μg/ml heparin, 1% antibiotic-antimycotic, and 15 μg/mL gentamycin at 37 °C in an atmosphere of 5% CO2. The number and volume of the mammospheres were determined under an Olympus IX 71 inverted microscope.
The matrix metalloproteinase MMP-2 and MMP-9 levels in mouse serum were measured using ELISA kits (R&D systems, Minneapolis, MN), according to the manufacturer's instructions. The quantity of MMP-2 and MMP-9 was determined by measuring the absorbance at 450 nm with a Spectramax MAX 190 microplate reader (Molecular Devices, CA).
2.13. Statistical analysis
All data were analyzed using GraphPad Prism 5.0 statistical soft-ware (San Diego, CA). The results are presented as mean ± SEM of at least three independent experiments. Data were analyzed by student's t-test, and one- or two-way ANOVA as appropriate. Significance between multiple experimental groups was determined using the Bonferroni post hoc test and defined at p* < 0.05.
3.1. L80 exerts cytotoxic effects on TNBC cells in vitro
We have previously synthesized the C-ring truncated deguelin de-rivative L80 , (Fig. 1A). We first sought to examine the cytotoxic effect of L80 on cell viability and apoptosis in MDA-MB-231, 4T1, BT549 and Hs578T cells in vitro. Following exposure to L80 (5 μM) for 72 h, TNBC cells exhibited significant morphological changes with concomitant cytoplasmic shrinkage and cellular rounding (Fig. 1B). MTS assays revealed that L80 (0.2–20 μM, 48–72 h) significantly sup-pressed cell viability in TNBC cells in a dose- and time-dependent manner (p < 0.05, Fig. 1C).