Journal: Journal of Medicinal Chemistry

Article Title: A novel targeted system to deliver chemotherapeutic drugs to EphA2-expressing cancer cells

doi: 10.1021/jm201743s

Figure Lengend Snippet: (A) EphA2 and EphA4 receptor expression in normal and cancer cell lines. Bar graph shows the fold expression of both EphA2 and EphA4 receptors in MDA-MD-231, PC-3, and LNCaP cancer cell lines compared to cultured human dermal fibroblast (HDF) and to a total human prostate RNA sample (Prostate). (B) Correlation between EphA2 mRNA expression and EphA2 protein levels in 30 selected human cancer cell lines. (C) Box and Whisker plots of the 10–90th percentile of EphA2 expression for the RNA samples grouped according to their tissue of origin. Dotted lines depict cutoffs for 7 fold, and 14 fold the expression levels observed in the reference HDF cell line, respectively.

Article Snippet: EphA2 Protein Quantification The amount of EphA2 protein in each cell line was assayed using the Human Total EphA2 DuoSet Elisa kit (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Cell Culture, Whisker Assay

Journal: Journal of Medicinal Chemistry

Article Title: A novel targeted system to deliver chemotherapeutic drugs to EphA2-expressing cancer cells

doi: 10.1021/jm201743s

Figure Lengend Snippet: (A) The YSA peptide targets quantum dots to EphA2 on the cell surface. COS cells transfected with either the extracellular and transmembrane portions of EphA2 fused to enhanced green fluorescent protein (EphA2-EGFP) or membrane targeted EGFP-F were incubated with YSA bound to red fluorescent quantum dots (YSA-Qdots). YSA-Qdots only bind to cells transfected with EphA2-EGFP. EGFP fluorescence is green; nuclei are stained in blue with DAPI. (B) YSA targets quantum dots to endogenous EphA2 on the cell surface. HUVE cells were incubated at 4ºC with YSA conjugated Qdots (YSA-Qdots), an unrelated 12-mer control peptide that does not bind to EphA2 also conjugated to Qdots (Ctrl-Qdots), or unconjugated Qdots (Qdots). Only quantum dots conjugated to the YSA peptide bind to the HUVE cells. (C) YSA targets Qdots to lysosomes. MDA-MB-231 cells, which express high levels of endogenous EphA2, were incubated at 4ºC with YSA-Qdots followed by incubation at 37 ºC for 0, 15, and 60 min. YSA-Qdots are seen on the cell surface at 0 min but become concentrated in structures near the nucleus at 15 and 60 min (arrow). Double labeling with the lysosomal marker Lamp1 (green) shows colocalization of the quantum dots in lysosomes at 60 min (arrows).

Article Snippet: EphA2 Protein Quantification The amount of EphA2 protein in each cell line was assayed using the Human Total EphA2 DuoSet Elisa kit (R&D Systems, Minneapolis, MN).

Techniques: Transfection, Membrane, Incubation, Fluorescence, Staining, Control, Labeling, Marker

Journal: Journal of Medicinal Chemistry

Article Title: A novel targeted system to deliver chemotherapeutic drugs to EphA2-expressing cancer cells

doi: 10.1021/jm201743s

Figure Lengend Snippet: (A) General chemical structures of YSA and DYP peptide-drug conjugates. (B) YSA, YSA-PTX and control DYP-PTX were incubated at the indicated concentrations together with a constant concentration of ephrin-A5 AP in ELISA wells pre-coated with EphA2 Fc. The ratio of ephrin-A5 AP bound in the presence and in the absence of peptide is shown. (C) Biotinylated YSA-PTX and control DYP-PTX were incubated at the indicated concentrations in EphA2 Fc-coated ELISA wells and were then detected with streptavidin-HRP. The graphs show averages ± SE from quadruplicate measurements in representative experiments, while the KD and IC50 values are calculated from 3 to 5 experiments. (D) ATP-Lite analysis of viability of PC-3 cells 72 hours after the addition of DYP, DYP-PTX, YSA, YSA-PTX or PTX at 10 nM (n = 3).

Article Snippet: EphA2 Protein Quantification The amount of EphA2 protein in each cell line was assayed using the Human Total EphA2 DuoSet Elisa kit (R&D Systems, Minneapolis, MN).

Techniques: Control, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Medicinal Chemistry

Article Title: A novel targeted system to deliver chemotherapeutic drugs to EphA2-expressing cancer cells

doi: 10.1021/jm201743s

Figure Lengend Snippet: EphA2 inhibition

Article Snippet: EphA2 Protein Quantification The amount of EphA2 protein in each cell line was assayed using the Human Total EphA2 DuoSet Elisa kit (R&D Systems, Minneapolis, MN).

Techniques: Sequencing

Journal: Journal of Medicinal Chemistry

Article Title: A novel targeted system to deliver chemotherapeutic drugs to EphA2-expressing cancer cells

doi: 10.1021/jm201743s

Figure Lengend Snippet: (A) YSA-PTX coupled to fluorescent quantum dots, but not DYP-PTX, is internalized with EphA2 into lysosomes of prostate cancer cells expressing EphA2. PC3 prostate cancer cells, which express high levels of EphA2, or LNCaP prostate cancer cells, which do not detectably express EphA2 protein, were treated for 20 min with 100 μM YSA-PTX or control DYP-PTX, followed by a 20 min incubation with streptavidin-conjugated quantum dots (Qdots). After removing the solution containing the peptides and the quantum dots, the cells were incubated for 2 h at 37°C to allow EphA2 internalization induced by YSA-PTX binding. The cells were stained for EphA2 (left) or the lysosomal marker Lamp1 (right). Qdots were also imaged and nuclei were labelled with DAPI in blue. Representative fluorescence micrographs are shown. Scale bar = 25 μM. (B) Ephrin-A1 Fc and YSA-PTX, but not DYP-PTX, causes EphA2 internalization into lysosomes. PC3 cells were treated for 2 h with 0.2 μg/mL ephrin-A1 Fc, 100 μM YSA-PTX or 100 μM control DYP-PTX. The cells were stained for Lamp1 (red) and EphA2 (green). Nuclei were labelled with DAPI (blue). Representative confocal micrographs are shown. Scale bar = 25 μm. (C) Ephrin-A1 Fc and YSA-PTX, but not DYP-PTX, cause EphA2 internalization into cells. PC3 prostate cancer and HUVE endothelial cells were treated for 1 h with 0.2 μg/mL ephrin-A1 Fc, 100 μM YSA-PTX or 100 μM control DYP-PTX. Proteins present on the cell surface were then labeled with biotin. EphA2 immunoprecipitates were probed with anti-phosphotyrosine antibody (PTyr), streptavidin-HRP (biotin) and EphA2 antibody. The amount of GAPDH in the cell lysates used for the immune-precipitations is also shown as a control for equal amounts of protein in the lysates used for immunoprecipitation.

Article Snippet: EphA2 Protein Quantification The amount of EphA2 protein in each cell line was assayed using the Human Total EphA2 DuoSet Elisa kit (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Control, Incubation, Binding Assay, Staining, Marker, Fluorescence, Labeling, Immunoprecipitation