Table 1.
Parameters used for bioinformatics analyses on kmplot.com.
Fig 1.
Kaplan-Meier analysis on SRC–3 level and survival rate of TNBC patients.
(A) Overall survival in low SRC–3 expression group and high SRC–3 expression group. (B) Overall survival in systemically untreated TNBC patients with low or high SRC–3 expression. (C) Distant metastasis-free survival in low or high SRC–3 expression group. (D) Post-progression survival in low or high SRC–3 expression group. KM-plotter database developed by Gyorffy et al. was used for analyses (51). Gene symbol: NCOA3. SRC–3 JetSet probe was used (Affymetrix ID: 209061_at). Patients were split by auto select best cutoff. Patients with basal-like intrinsic subtype was selected. For statistical significance, Log Rank test was used.
Fig 2.
Bufalin downregulates SRC–3 and decreases cell viability in TNBC cells.
(A) Western blot shows that bufalin (100 nM) downregulates SRC–3 protein levels in TNBC cells. IC50 values, TNBC subtypes and gene ontologies are also listed. (B) Western blotting shows a dose-dependent downregulation of SRC–3 by bufalin and phospho-bufalin in LM3-3 cells. (C-H) Bufalin decrease cell viability of TNBC cells including HCC1143 (C), SUM149PT (D), SUM159PT (E), MDA-MB–231 (F) and MDA-MB-231-LM3-3 (G), but not in primary mouse hepatocytes (H). Data are presented as mean±SD.
Fig 3.
Bufalin downregulates SRC–3 and reduces cell motility in MDA-MB-231-LM3-3.
(A) Cell motility assay of LM3-3 cells was performed using a Cellomics cell motility kit. The bright areas of 50 images for each sample were analyzed using Image J. (B) Treatment of LM3-3 cells with different concentrations of bufalin for 12 h showed minimal toxicity but significant motility reduction. Data are presented as mean±SD.
Fig 4.
Synergistic effect of Gefitinib and bufalin in TNBC cells.
When combined with 5 or 10 nM of bufalin, co-treatment with the EGFR inhibitor gefitinib synergistically reduced LM3-3 cell viability. Data are presented as mean±SD.
Fig 5.
Synthetic scheme of 3-phospho-bufalin.
Table 2.
Electrocardiogram (EKG) measurements in mice treated with bufalin and 3-phospho-bufalin.
Fig 6.
H-E staining of bufalin or 3-phospho-bufalin treated mouse heart.
H&E staining of heart tissues 24 h after drug administration. Neither of bufalin or 3-phospho-bufalin caused severe damage to the heart muscle, indicating that the acute toxicity of bufalin is reversible.
Fig 7.
Pharmacokinetics (PK) of 3-Phospho-bufalin (p-Buf).
PK trace of p-buf (orange) and free bufalin generated from p-buf (blue) in mice treated with p-buf (0.5 mg/kg). The blue and orange dotted lines represent the lower limit of quantification (LLOQ) for bufalin and p-buf in this assay, respectively.
Table 3.
Pharmacokinetic (PK) parameters of 3-Phospho-bufalin.
Fig 8.
Therapeutic efficacy of 3-phospho-bufalin in an orthotopic TNBC model.
(A) LM3-3 cells were inoculated into the mammary fat pads of nude mice (female, 4–5 weeks). The treatment was started 14 days after tumor inoculation. The treatment group was treated with 3-phospho-bufalin (0.75 mg/kg) 3 times per week for 3 weeks (n = 6). The control group was treated with PBS (n = 6). Tumor volumes was measured three times per week. As shown, 3-phospho-bufalin can significant inhibit TNBC tumor growth. Data are presented as mean±SEM. *, P < 0.05; **, P< 0.01, by t-test. (B) Representative images of harvested tumors. (C) Comparison of the tumor weights from both the 3-phospho-bufalin treated group and the PBS treated control group. Data are presented in a box plot with mean, minimum and maximum values. P = 0.0156 by t-test. (D) Body weight of mice at the end of the experiment.(E) Western blotting analyses of the SRC–3 protein levels in the tumor tissues of the p-Buf and PBS treated mice.
Fig 9.
P-buf downregulates SRC–3 levels and reduces the number of Ki–67 positive cells in an orthotopic TNBC model.
Multiple tumor tissues (N>3) collected from both control group (administrated with PBS), and p-buf treated group were processed and immunohistochemically stained with anti-SRC–3 antibody (upper panel), or anti-Ki–67 antibody (bottom panel). The staining results were photographed under a microscope. One representative photo for each staining is presented.