cb 2 r-selective antagonist sr144528 sr2  (Tocris)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Therapeutic targeting of HER2–CB 2 R heteromers in HER2-positive breast cancer

doi: 10.1073/pnas.1815034116

Figure Lengend Snippet: THC decreases HER2–CB2R complexes. (A) Schematic representation of bioluminescence resonance energy transfer experiments. (B) BRET saturation curve in HEK293 cells transfected with a fixed concentration of HER2-Rluc and increasing concentrations of CB2R-YFP. HER2-Rluc/GHS-R1a-YFP and D44R-Rluc/YFP were used as negative controls for the interaction (n = 8). (C) Effect of THC (4 h), alone or in combination with the CB2R-selective antagonist SR144528 (SR2; 1 µM), on HER2-Rluc/CB2R-YFP BRETmax signal in HEK293 cells (n = 3). (D and E) Viability of CB2R- and HER2-transfected HEK293 cells after 24-h treatment with increasing concentrations of THC (n = 5) (D), or THC in combination with SR2 (1 μM) (n = 4) (E). (F and G) Viability of BT474 (n = 6) and HCC1954 (n = 3) cells in response to increasing concentrations of THC (F), or in combination with the CB2R-selective antagonist SR144528 (1 μM) (G). Results (n = 3 to 6 independent experiments) are expressed as percent vs. vehicle-treated cells, set at 100%, and error bars represent SEM. (H) Coimmunoprecipitation of HER2 with CB2R after THC treatment (4 h), in BT474 and HCC1954 cells transfected with an HA-tagged CB2R plasmid. IP, immunoprecipitation. (I) Representative PLA confocal microscopy images of HER2–CB2R heteromers (in red) in BT474 (Upper) and HCC1954 cells (Lower), treated with THC (4 h) alone or in combination with SR2 (1 μM). Cell nuclei are stained in blue. (Scale bars, 25 µm.) (J) Quantification of HER2–CB2R PLA signal (number of red dots per cell) (n = 3). Results are expressed as percent vs. vehicle-treated cells, set at 100%, and error bars represent SEM. Multigroup comparisons were analyzed by one-way ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01 vs. vehicle-treated cells; # P < 0.05, ## P < 0.01 vs. THC.

Article Snippet: The CB 2 R-selective antagonist SR144528 (SR2) (Tocris Bioscience) and lactacystin (Calbiochem) were dissolved in DMSO and added to the cell cultures (1 μM) 1 h prior to THC.

Techniques: Bioluminescence Resonance Energy Transfer, Transfection, Concentration Assay, Plasmid Preparation, Immunoprecipitation, Confocal Microscopy, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Therapeutic targeting of HER2–CB 2 R heteromers in HER2-positive breast cancer

doi: 10.1073/pnas.1815034116

Figure Lengend Snippet: HER2–CB2R heteromer disruption by THC hampers HER2 activation. (A) HER1, HER2, HER3, and HER4 expression, as determined by Western blot analysis, in the indicated breast cancer cell lines. (B) Representative PLA confocal microscopy images of the effect of THC (4 h) on HER2–HER1 (n = 4), HER2–HER2 (n = 5), and HER2–HER3 (n = 3) dimers (in red) in HCC1954 cells (B), with the corresponding quantification (C), or on HER2–HER2 expression after THC treatment, alone or in combination with the CB2R-selective antagonist SR144528 (1 μM) (n = 3) (D), with the corresponding quantification (E). Cell nuclei are in blue. (Scale bars, 20 µm.) (F and G, Left) Schematic representation of the BRET experiments conducted in HEK293 cells. CoH, coelenterazine H. (F and G, Right) Quantification of HER2-Rluc/HER2-YFP BRETmax after THC treatment (4 h) alone or in combination with SR2 (1 µM) where indicated, in cells cotransfected with HER2-Rluc, HER2-YFP, and a CB2R untagged receptor (n = 3) (F), or an empty vector (n = 4) (G) (used as a negative control for THC activation). In C and E–G, results are expressed as percent vs. vehicle-treated cells, set as 100%, and graph bars represent SEM. (H) Expression of pHER21248 in BT474 and HCC1954 cells, as determined by Western blot, upon THC treatment at the indicated times. (I) Quantification. Results are normalized vs. the corresponding total HER2 levels at each individual time point, and expressed as fold increase vs. time 0, set at 1 (n = 4 in BT474; n = 7 in HCC1954). Error bars represent SEM. Unpaired independent groups of two were analyzed by two-tailed Student’s t test. When multigroup comparison was required, data were analyzed by one-way ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01 vs. vehicle-treated cells; ## P < 0.01 vs. THC. n.s., not significant.

Article Snippet: The CB 2 R-selective antagonist SR144528 (SR2) (Tocris Bioscience) and lactacystin (Calbiochem) were dissolved in DMSO and added to the cell cultures (1 μM) 1 h prior to THC.

Techniques: Disruption, Activation Assay, Expressing, Western Blot, Confocal Microscopy, Plasmid Preparation, Negative Control, Two Tailed Test, Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Therapeutic targeting of HER2–CB 2 R heteromers in HER2-positive breast cancer

doi: 10.1073/pnas.1815034116

Figure Lengend Snippet: HER2–CB2R heteromer disruption by THC induces HER2 degradation in vitro and in vivo. (A, B, and D) Effect of THC on HER2 protein (A and B) and mRNA levels (D) at the indicated times, as determined by Western blot and qPCR, respectively, in BT474 and HCC1954 cells. For quantification, HER2 expression was normalized with the loading control [β-actin in B; β-actin and β-glucuronidase in D], and results (n = 4 in B; n = 3 in D) are expressed as fold increase vs. time 0, set at 1. Data were analyzed by one-way ANOVA. (C) Western blot analysis of the effect of the CB2R-selective antagonist SR144528 (1 μM) on THC-induced HER2 protein decrease (n = 4 in BT474; n = 7 in HCC1954). (E) Growth of orthotopic tumors generated in NOD-SCID mice by injection of HCC1954 cells into the mammary fat pad. Animals were treated with vehicle (sesame oil) (n = 10) or THC (1.5 mg per dose) (n = 9) thrice a week. Results were analyzed by two-way ANOVA. (F) Representative Western blot of HER2 in the animal tumor samples. (G) Corresponding quantification. (H) Representative PLA confocal microscopy images of HER2–CB2R and HER2–HER2 heteromers (red signal). Cell nuclei are in blue. (Scale bar, 50 µm.) (I) Quantification. Error bars in B, D, E, and I represent SEM. Unpaired, two-tailed Student’s t test. *P < 0.05, **P < 0.01 vs. time 0 (B) or vehicle-treated animals in E, G, and I.

Article Snippet: The CB 2 R-selective antagonist SR144528 (SR2) (Tocris Bioscience) and lactacystin (Calbiochem) were dissolved in DMSO and added to the cell cultures (1 μM) 1 h prior to THC.

Techniques: Disruption, In Vitro, In Vivo, Western Blot, Expressing, Control, Generated, Injection, Confocal Microscopy, Two Tailed Test