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

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 via the c-CBL E3 ligase. Western blot-based analyses of the effect of different pharmacological and genetic tools on THC-induced HER2 degradation. (A and B) Effect of lactacystin (LAC; 1 µM) on BT474 cells (n = 4). (C–F) Effect of THC (4 h) on ubiquitinated HER2 (UB) (C), on c-CBL and CHIP levels (D and E), or on HER2 phosphorylation at Tyr1112 (F) in the indicated breast cancer cell lines. (G and H) HER2 protein expression after genetic silencing of c-CBL with selective siRNAs (siCBL). A nontargeted siRNA was used as a control (siC). The densitometric analyses of HER2 immunoblots were normalized to β-actin (n = 4 in B; n = 6 in E; n = 4 in H). Results are expressed as fold increase vs. vehicle-treated cells, set at 1, and graph 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 group; # P < 0.05, ## P < 0.01 vs. THC-treated group.

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, Western Blot, Phospho-proteomics, Expressing, Control, Two Tailed Test, Comparison

Journal: Molecular Pain

Article Title: Anti-nociceptive interactions between opioids and a cannabinoid receptor 2 agonist in inflammatory pain

doi: 10.1177/1744806917728227

Figure Lengend Snippet: JWH-133 acts through CB2R. The efficacy of JWH-133 (1 mg/kg, i.p.) in the formalin test was examined in the presence of CB1R and CB2R (a and b), as well as MOR (c and d) antagonists administered 30 min prior to JWH-133 treatment ( n = 4–5 per treatment.) The anti-nociceptive effect of JWH-133 was blocked by CB2R antagonist SR2 (10 mg/kg, i.p.) in both acute (a) and inflammatory pain (b). However, JWH-133 was not impacted by the CB1R antagonist SR1 (10 mg/kg; a and b) or by MOR antagonist naloxone (10 mg/kg; c and d). * p < 0.046 for JWH-133 and versus vehicle group (ANOVA with Bonferroni post hoc). ANOVA: analysis of variance; CB1R: cannabinoid 1 receptor; CB2R: cannabinoid 2 receptor; MOR: mu-opioid receptor; AUC: area under the curve.

Article Snippet: JWH-133 (CB2R agonist), SR144528 (SR2, CB2R antagonist), naloxone (MOR antagonist), and Rimonabant (SR1, CB1R antagonist) were obtained from Cayman Chemical (Ann Arbor, MI).

Techniques:

Journal: Molecular Pain

Article Title: Anti-nociceptive interactions between opioids and a cannabinoid receptor 2 agonist in inflammatory pain

doi: 10.1177/1744806917728227

Figure Lengend Snippet: Lack of JWH-133-induced adverse effects. Mice ( n = 5–7 per group) were injected with a maximal dose of JWH-133 (1 mg/kg), or morphine (10 mg/kg) for seven days to look for either acute or cumulative physical side effects. The hypothermic effects of JWH-133 were not significantly different from vehicle according to a two-way ANOVA with Dunnett’s post hoc test (a). The same trends were observed when measuring the effect of JWH-133 on motor impairment using a rotarod apparatus (b). Comparatively, morphine-injected mice showed significant hypothermic effects on day 1 (* p = 0.001), but not day 4 ( p = 0.1334), or day 7 ( p = 0.1673). Morphine also caused significant motor impairment on day 1 (* p = 0.001), but not day 4 ( p = 0.2120), or day 7 ( p = 0.8429). Following 10 days of twice-daily drug administration, animals were administered antagonists, and frequency of physical withdrawal behaviors were recorded for 60 min (c). While SR2 administration following repeated JWH-133 did not result in withdrawal behavior, morphine-treated mice challenged with naloxone showed significant amounts of paw tremors (*** p < 0.001), body tremors (*** p < 0.001), and jumping (*** p < 0.001) relative to vehicle in a one-way ANOVA. ANOVA: analysis of variance.

Article Snippet: JWH-133 (CB2R agonist), SR144528 (SR2, CB2R antagonist), naloxone (MOR antagonist), and Rimonabant (SR1, CB1R antagonist) were obtained from Cayman Chemical (Ann Arbor, MI).

Techniques: Injection

Journal: Molecular Pain

Article Title: Anti-nociceptive interactions between opioids and a cannabinoid receptor 2 agonist in inflammatory pain

doi: 10.1177/1744806917728227

Figure Lengend Snippet: Cross-tolerance between JWH-133 and morphine. The formalin model of inflammatory pain was utilized to examine cross-tolerance between morphine and JWH-133. The composite pain score (a) and AUC for acute (b) and inflammatory pain (c) is shown for male mice that received a challenge dose of morphine (10 mg/kg, i.p.) after 10 days of repeated JWH-133 (1 mg/kg, i.p.) administration. A second group received a challenge dose of JWH-133 following repeated morphine and is displayed as a composite pain score (d) and AUC for acute (e) and inflammatory pain (f). A group of female mice also received a challenge dose of JWH-133 following repeated morphine and are displayed as AUC for acute (g) and inflammatory pain (h). Data are expressed as mean ± SEM ( n = 5–8 per group). * p < 0.0001 versus control group (ANOVA with Bonferroni post hoc); + p < 0.01 versus control or repeated morphine + JWH-133 challenge groups (ANOVA with Bonferroni post hoc). ANOVA: analysis of variance; AUC: area under the curve; SEM: standard error of the mean.

Article Snippet: JWH-133 (CB2R agonist), SR144528 (SR2, CB2R antagonist), naloxone (MOR antagonist), and Rimonabant (SR1, CB1R antagonist) were obtained from Cayman Chemical (Ann Arbor, MI).

Techniques:

Journal: Molecular Pain

Article Title: Anti-nociceptive interactions between opioids and a cannabinoid receptor 2 agonist in inflammatory pain

doi: 10.1177/1744806917728227

Figure Lengend Snippet: Anti-nociceptive efficacy of JWH-133. Wild-type male (a) and female (b) mice ( N = 4–7 per dose) were tested using the formalin model. Testing was conducted 60 min after i.p. injection of JWH-133 (0.01, 0.03, 0.1, 0.3, 1, 3, 10 mg/kg). The area under the curve (AUC) represents the pain behavior obtained from a composite pain score. Treatment with JWH-133 reduced both acute ( F (6, 35) = 16.02, p < 0.0001) and inflammatory ( F (6, 35) = 21.53, p < 0.0001) pain in a dose-dependent fashion, with a maximal effect occurring at a dose of 1 mg/kg. The calculated ED 50 values for acute and inflammatory phases were 0.2295 mg/kg and 0.2294 mg/kg for males. For females, the ED 50 values were 0.2431 mg/kg and 0.2030 mg/kg.

Article Snippet: JWH-133 (CB2R agonist), SR144528 (SR2, CB2R antagonist), naloxone (MOR antagonist), and Rimonabant (SR1, CB1R antagonist) were obtained from Cayman Chemical (Ann Arbor, MI).

Techniques: Injection

Journal: Molecular Pain

Article Title: Anti-nociceptive interactions between opioids and a cannabinoid receptor 2 agonist in inflammatory pain

doi: 10.1177/1744806917728227

Figure Lengend Snippet: Comparison of morphine and JWH-133 in the formalin test. Mice ( n = 4–6 per dose) were tested in the formalin model 60 min after i.p. administration of maximal doses of either JWH-133 (1 mg/kg) or morphine (10 mg/kg). The area under the curve (AUC) represents the pain behavior obtained from the composite pain score. Both morphine and JWH-133 reduced pain behavior relative to the vehicle group in the acute (a) and inflammatory (b) phases. Morphine produced a greater anti-nociceptive effect than JWH-133 in both phases. * p < 0.0001 for JWH-133 and morphine versus vehicle group (ANOVA with Bonferroni post hoc) and + p < 0.037 for morphine versus JWH-133 group (ANOVA with Bonferroni post hoc). ANOVA: analysis of variance.

Article Snippet: JWH-133 (CB2R agonist), SR144528 (SR2, CB2R antagonist), naloxone (MOR antagonist), and Rimonabant (SR1, CB1R antagonist) were obtained from Cayman Chemical (Ann Arbor, MI).

Techniques: Produced

Journal: Molecular Pain

Article Title: Anti-nociceptive interactions between opioids and a cannabinoid receptor 2 agonist in inflammatory pain

doi: 10.1177/1744806917728227

Figure Lengend Snippet: Lack of observed tolerance to JWH-133. Mice ( n = 3–5 per group) were measured in the formalin test following 1, 6, or 11 days of JWH-133 (1 mg/kg; a) or morphine (10 mg/kg; b) administration. JWH-133 (a) and morphine (b) reduce pain behavior relative to vehicle group. This anti-nociceptive effect of JWH-133 is similar on all days examined. For morphine, a greater anti-nociceptive effect is observed at day 1 relative to day 6 and from day 6 relative to day 11. Data are expressed as mean ± SEM. * p < 0.0001 for JWH-133 or morphine versus vehicle group (ANOVA with Bonferroni post hoc); + p < 0.001 for morphine day 1 versus morphine day 6 group (ANOVA with Bonferroni post hoc); # p < 0.039 for morphine day 6 versus morphine day 11 group (ANOVA with Bonferroni post hoc). ANOVA: analysis of variance; AUC: area under the curve; SEM: standard error of the mean.

Article Snippet: JWH-133 (CB2R agonist), SR144528 (SR2, CB2R antagonist), naloxone (MOR antagonist), and Rimonabant (SR1, CB1R antagonist) were obtained from Cayman Chemical (Ann Arbor, MI).

Techniques:

Journal: Molecular Pain

Article Title: Anti-nociceptive interactions between opioids and a cannabinoid receptor 2 agonist in inflammatory pain

doi: 10.1177/1744806917728227

Figure Lengend Snippet: JWH-133 co-administration modestly protects against morphine tolerance. The efficacy of morphine (10 mg/kg) in the formalin model was compared between mice receiving daily morphine alone (10 mg/kg) and mice receiving daily morphine (10 mg/kg) and daily JWH-133 (1 mg/kg). Co-administration of JWH-133 with morphine resulted in significantly greater acute phase 1 (a) and phase 2 (b) anti-nociception on day 6 and day 11. Data are expressed as mean ± SEM ( n = 4 per group). * p < 0.004 versus vehicle group (ANOVA with Bonferroni post hoc); + p < 0.047 versus morphine + morphine challenge group (ANOVA with Bonferroni post hoc); # p < 0.003 versus combination + morphine challenge group (ANOVA with Bonferroni post hoc). ANOVA: analysis of variance; AUC: area under the curve; SEM: standard error of the mean.

Article Snippet: JWH-133 (CB2R agonist), SR144528 (SR2, CB2R antagonist), naloxone (MOR antagonist), and Rimonabant (SR1, CB1R antagonist) were obtained from Cayman Chemical (Ann Arbor, MI).

Techniques:

Journal: Molecular Pain

Article Title: Anti-nociceptive interactions between opioids and a cannabinoid receptor 2 agonist in inflammatory pain

doi: 10.1177/1744806917728227

Figure Lengend Snippet: JNK signaling is partially responsible for morphine-induced cross-tolerance to JWH-133. The efficacy of a challenge dose of JWH-133 (1 mg/kg) in the formalin model was compared between mice that received five days of daily morphine alone (10 mg/kg), mice receiving daily morphine (10 mg/kg) and SP6 (3 mg/kg), and mice receiving daily vehicle. Co-administration of SP6 with morphine resulted in a significant increase of JWH-133 efficacy both in acute and inflammatory pain. Data are expressed as mean ± SEM ( n = 3–5 per group). * p < 0.0001 versus chronic morphine group (ANOVA with Bonferroni post hoc); + p < 0.001 versus chronic morphine + SP6 group (ANOVA with Bonferroni post hoc). JNK: c-Jun N-terminal kinase; ANOVA: analysis of variance; AUC: area under the curve; SEM: standard error of the mean.

Article Snippet: JWH-133 (CB2R agonist), SR144528 (SR2, CB2R antagonist), naloxone (MOR antagonist), and Rimonabant (SR1, CB1R antagonist) were obtained from Cayman Chemical (Ann Arbor, MI).

Techniques:

Journal: Molecular Pain

Article Title: Anti-nociceptive interactions between opioids and a cannabinoid receptor 2 agonist in inflammatory pain

doi: 10.1177/1744806917728227

Figure Lengend Snippet: The anti-nociceptive effects of JWH-133 and morphine are additive in the formalin test. A dose–response curve was generated in the formalin test for co-administration of JWH-133 and morphine in a 1:10 fixed dose ratio ( n = 3–6 per dose). Non-linear isobolographic analysis was used to compare the experimentally determined ED 50 values with theoretical ED 50 values if the combination were exactly additive. In phase 1 (c), the theoretical ED 50 (0.7765 ± 0.18 mg/kg) was found to overlap with the experimentally determined value (0.7236 ± 0.11 mg/kg), suggesting additivity. In phase 2 (d) of the formalin test, the experimentally determined ED 50 (0.6211 ± 0.063 mg/kg) appears to be lower than the predicted ED 50 (0.9258 ± 0.12 mg/kg). However, the 95% confidence intervals of the two values are overlapping. AUC: area under the curve.

Article Snippet: JWH-133 (CB2R agonist), SR144528 (SR2, CB2R antagonist), naloxone (MOR antagonist), and Rimonabant (SR1, CB1R antagonist) were obtained from Cayman Chemical (Ann Arbor, MI).

Techniques: Generated