Fig 1: Evaluation of the combinatorial effect of Lupeol and 5FU on MDA-MB 231 cells(A) Western blot analysis of MDA-MB-231 cells treated with 5FU, Lupeol, or both in the presence of HGF and probed for phospho-EphA2 (S897), EphA2, phospho-c-MET, total c-MET, pERK1/2, total ERK1/2, MMP2 and Laminin-5Ƴ2 protein expression. β- Actin was used as a loading control.(B) Protein-protein interaction (PPI) network analysis using input proteins from the present study using STRING platform showing interacting molecules and edge confidence levels in the network.(C and D) Transwell migration and invasion of MDA-MB-231 cells upon treatment with Lupeol, 5FU or both, while HGF was used as a chemotactic factor (Scale bars: 100 μm).(E) Matrigel tube formation of MDA-MB-231 cells upon treatment with Lupeol, 5FU, or both in the presence of HGF (Scale bars: 500 μm and 100 μm in inset).(F) Primary and secondary Mammosphere formation of MDA-MB-231 cells in the presence of HGF and treated with 5FU, Lupeol, or both (Scale 200 μm in white and 100 μm in red).(G) Immunofluorescence staining of MDA-MB-231 cells for the detection of E-cadherin (green) and Vimentin (red) post treatment with Lupeol or 5FU or both in the presence of HGF (Scale bars: 100 μm).(H) Western blot analysis of MDA-MB-231 cells treated with Lupeol, 5FU, or both in the presence of HGF and probed for E-cadherin, Vimentin, SNAI1, SLUG, and TWIST. Data are representative of triplicate experiments (mean ± SD). HGF = hepatocyte growth factor; HF = HGF+5FU; HL = HGF+Lupeol; HFL = HGF+5FU + Lupeol. Also see, Figure S5.
Fig 2: In vivo evaluation of the combinatorial effect of Lupeol and 5FU in a TNBC syngeneic mice model(A) Schematic representation of dosing schedule (all compounds were administered intraperitoneally).(B) In vivo live animal images of the 4T1luc2 induced tumors in various treatment arms in BALB/c mice at day 5 (initiation of treatment) and at day 15 (experimentation endpoint).(C) Graph representing the log of average radiance vs. Time of the tumor growth.(D) Representative photomicrograph of freshly harvested breast tumors with a ruler (below) for scale.(E) Graph representing the tumor volume vs. the various treatment arms.(F) Representative images of Hematoxylin and Eosin stain followed by IHC staining for Ki67 and Caspase 3c in the sections of harvested tumors developed in BALB/c mice along with their quantitative graphs.(G) Representative images of the IHC staining to evaluate the differential expression of phospho-EphA2 and phospho-cMET in the tumors of the various treatment arms along with their quantitative graphs. ∗p < 0.05 and ∗∗∗p < 0.001 statistically significant difference compared to corresponding control by one-way ANOVA. Data are representative of triplicate experiments (mean ± SD) ns = not significant; HGF = hepatocyte growth factor; HF = HGF+5FU; HL = HGF+Lupeol; HFL = HGF+5FU + Lupeol. Scale bars: 200 μm. Also see, Figure S6 and Table S8.
Fig 3: Evaluation of the effect of individual and dual knock down of c-MET and EphA2 genes on MD-MB-231 in the presence of HGF(A) c-MET and EphA2 knockdown/inhibition and its effect on downstream effectors on MDA-MB-231 cells was analyzed by western blot; NTP = non-target pool control.(B) Transwell migration assay post knockdown/inhibition of c-MET or EphA2 or both and HGF as a chemotactic factor on MDA-MB-231 cells.(C) Transwell invasion assay post knockdown/inhibition of EphA2 and c-MET or both and HGF as a chemotactic factor on MDA-MB-231 cells.(D) A schematic representation of the mammosphere formation assay.(E) Mammosphere formation assay post knockdown/inhibition of c-MET or EphA2 or both in the presence of HGF.(F) Matrigel tube formation assay post knockdown/inhibition of c-MET or EphA2 or both in the presence of HGF.(G) Representative photomicrograph of freshly harvested breast tumors with a ruler (below) for scale.(H) Graph representing the tumor volume vs. the various groups. Data are representative of triplicate experiments (mean ± SD). ∗p < 0.05 statistically significant difference compared to corresponding control by one-way ANOVA. Scale bars: 100 μm. Also see, Figures S3 and S4.
Fig 4: Combination of Lupeol and 5FU induces cytotoxicity on MDA-MB-231 cells and also reduce their wound healing potential(A) Percentage cell viability graph depicting the various Combination treatment of MDA-MB-231 cells with Lupeol (0–20 μM) and 5FU (0, 5, 10, and 15 μM).(B) Dose response heatmap based on percentage inhibition of cell viability following combination of Lupeol (0–20 μM) and 5FU (0–15 μM) in MDA-MB-231 cells. Scale represents the spectrum where increase in the intensity of red color indicates higher percentage of inhibition.(C) The synergy distribution map of the combination of Lupeol and 5FU using Bliss synergy model that calculated Bliss synergy score.(D) Colony formation assay post treatment with Lupeol (8 μM) and/or 5FU (10 μM) of MDA-MB-231 cells (in the presence of HGF).(E) Graph representing the relative cell plating efficiency (%) of MDA-MB-231.(F) Wound healing assay post treatment with Lupeol (8 μM) and/or 5FU (10 μM) on MDA-MB-231 cells in the presence of HGF (100 ng/mL) for 24 and 48 h.(G) Graph representing the relative wound area (%) over 24 and 48 h. Data are representative of triplicate experiments (mean ± SD). ∗p < 0.05 and ∗∗∗p < 0.001 statistically significant difference compared to corresponding control by one-way ANOVA. ns = not significant; HGF = hepatocyte growth factor; HF = HGF+5FU; HL = HGF+Lupeol; HFL = HGF+5FU + Lupeol. Scale bars: 500 μm. Also see, Table S7.
Fig 5: Evaluation of the combinatorial effect of 5FU and Lupeol in ex vivo explant culture model(A) Graphical representation of the patient data used in this experiment, pertaining to the nodal status and tumor stage.(B) Representative images of IHC staining of TNBC tissue fragments, post exposure to HGF, 5FU, Lupeol alone or in combination cells for the detection of Ki67 and Caspase 3c protein expression (Scale bars: 200 μm).(C) Graph representing the percentage of cells expressing Ki67 vs. the various treatment arms.(D) Graph representing the IHC score of Caspase 3c vs. various treatment arms.(E) Representative images of IHC staining for the evaluation of phospho-EphA2 and phospho-cMET in various treatment arms (Scale bars: 200 μm and 100 μm in inset).(F) Graph representing the various IHC scores of phospho-EphA2 in various treatment arms.(G) Graph representing the various IHC scores of phospho-cMET in various treatment arms.(H) Graph representing the various IHC scores of Caspase 3c expression in TNBC tumors (N = 7) after treating them with various SOC and 5FU and Lupeol combination. Data are representative of triplicate experiments (mean ± SD). ∗p < 0.05 statistically significant difference compared to corresponding control by one-way ANOVA. HGF = hepatocyte growth factor; HF = HGF+5FU; HL = HGF+Lupeol; HFL = HGF+5FU + Lupeol. Also see, Table S9.
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