Journal: Cells

Article Title: Functional Selectivity of Cannabinoid Type 1 G Protein-Coupled Receptor Agonists in Transactivating Glycosylated Receptors on Cancer Cells to Induce Epithelial–Mesenchymal Transition Metastatic Phenotype

doi: 10.3390/cells13060480

Figure Lengend Snippet: Sialidase activity is associated with CB1 GPCR agonist treatments of live RAW-blue macrophage cells. ( A ) We propose that the CB1 GPCRs heterodimerize with neuromedin B (NMBR) tethered to TLR in a multimeric receptor complex with matrix metalloproteinase-9 (MMP-9) and neuraminidase-1 (Neu1) in naïve (unstimulated) TLR-expressing cells. Here, a novel molecular signaling platform regulating the interaction and signaling mechanism between these molecules on the cell surface uncovers a functional selectivity of CB1 GPCR biased heteromers with NMBR to induce TLR activation signaling axis mediated by Neu1 sialidase activation and the modification of TLR glycosylation. This signaling platform potentiates MMP9 and Neu1 crosstalk on the cell surface, which is essential for activating TLR. Citation: Taken in part from Bunsick et al. . ( B ) Cells were allowed to adhere on 12 mm circular glass slides for 24 h at 37 °C in a humidified incubator. After removing the media, 0.318 mM 4-MUNANA substrate in Tris-buffered saline pH 7.4 was added to cells alone (control background) or with either AM-404, Arvanil, and Olvanil at the indicated dosage. Fluorescent images were taken 2 min after adding substrate using epi-fluorescent microscopy (20× objective). The sialidase hydrolyzed product of 4-MUNANA (4-MU) has an emission at 450 nm (blue color) when excited at 365 nm. The mean fluorescence of 50 multi-point replicates surrounding the live cells’ periphery was calculated using Image J software, 1.5g, Java 1.8.0_345 (64-bit). The mean fluorescence ± S.E.M is represented by error bars. ( C ) Different components of the signaling paradigm were inhibited using BIM-23127, an antagonist of NMBR, MMP9i, an inhibitor of MMP-9, and oseltamivir phosphate (OP), an inhibitor of Neu-1 at the indicated predetermined concentrations. The quantified data represent two to three independent experiments displaying similar results. Statistical significance, as indicated by asterisks, was calculated with ANOVA and Fisher’s LSD uncorrected multiple comparisons post hoc test at a confidence level of 95%. ns = non-significant, **** p < 0.0001, * p < 0.05.

Article Snippet: The CB1 agonists, N-arachidonoyl aminophenol (AM 404) (KI: 2.57 μg/mL) [ ], Arvanil (KI: 1.28 μg/mL) [ ], and Olvanil (KI: 4.127 μg/mL) [ ], were purchased from Alomone Labs, Jerusalem BioPark (JBP) (Jerusalem, Israel) and utilized in a dose-dependent manner.

Techniques: Activity Assay, Expressing, Functional Assay, Activation Assay, Modification, Saline, Control, Microscopy, Fluorescence, Software

Journal: Cells

Article Title: Functional Selectivity of Cannabinoid Type 1 G Protein-Coupled Receptor Agonists in Transactivating Glycosylated Receptors on Cancer Cells to Induce Epithelial–Mesenchymal Transition Metastatic Phenotype

doi: 10.3390/cells13060480

Figure Lengend Snippet: ( A ) We propose that the CB1 GPCRs heterodimerize with neuromedin B (NMBR) tethered to RTK in a multimeric receptor complex with matrix metalloproteinase-9 (MMP-9) and neuraminidase-1 (Neu1) in naïve (unstimulated) cancer cells. Citation: Taken in part from Bunsick et al. and Haxho et al. . Sialidase activity of live PANC-1 ( B , C ) and SW-620 ( D , E ) in response to AM-404, Arvanil, and Olvanil. CB1 agonists, AM-404, Arvanil, and Olvanil, significantly induce Neu-1 sialidase activity in a dose-dependent fashion compared to media control in live PANC-1 ( B ) and SW-620 ( D ). In the three cell lines, the saturation and KI concentrations for each agonist produced a more significant impact on sialidase activity compared to the latter two concentrations, which were more representative of the control. The sialidase hydrolyzed product of 4-MUNANA (4-MU) has an emission at 450 nm (blue color) when excited at 365 nm. The mean fluorescence of 50 multi-point replicates surrounding the cell’s periphery was calculated using Image J software. The mean fluorescence ± S.E.M is represented by error bars. ( C , E ) Different components of the signaling paradigm were inhibited using BIM-23127, an antagonist of NMBR, MMP9i, an inhibitor of MMP-9, and oseltamivir phosphate (OP), an inhibitor of Neu-1 at the indicated predetermined concentrations. The quantified data represent two to three independent experiments displaying similar results. Statistical significance, as indicated by asterisks, was calculated with ANOVA and Fisher’s uncorrected LSD multiple comparisons post hoc test at a confidence level of 95%. ns = non-significant, **** p < 0.0001, ** p < 0.01.

Article Snippet: The CB1 agonists, N-arachidonoyl aminophenol (AM 404) (KI: 2.57 μg/mL) [ ], Arvanil (KI: 1.28 μg/mL) [ ], and Olvanil (KI: 4.127 μg/mL) [ ], were purchased from Alomone Labs, Jerusalem BioPark (JBP) (Jerusalem, Israel) and utilized in a dose-dependent manner.

Techniques: Activity Assay, Control, Produced, Fluorescence, Software

Journal: Cells

Article Title: Functional Selectivity of Cannabinoid Type 1 G Protein-Coupled Receptor Agonists in Transactivating Glycosylated Receptors on Cancer Cells to Induce Epithelial–Mesenchymal Transition Metastatic Phenotype

doi: 10.3390/cells13060480

Figure Lengend Snippet: Immunofluorescence analysis of E-cadherin expression in response to CB1-agonists. CB1-agonists, AM-404, Arvanil, and Olvanil notably reduced the expression of the epithelial–mesenchymal transition (EMT), E-cadherin, in SW-620 cells. Cells were fixed, permeabilized, blocked, and immunostained with primary mouse monoclonal IgG antibodies for E-cadherin, followed by goat anti-mouse AlexaFluor 488 (green) secondary antibodies. As an isotype control, we use normal mouse IgG. The fluorescence was calculated using Corel Photo-Paint, with the average of eight points subtracted by the background fluorescence and multiplied by the pixel density (20× objective). Statistical significance was calculated with ANOVA and Fisher’s uncorrected LSD multiple comparisons post hoc test at a confidence level of 95%. ns = non-significant.

Article Snippet: The CB1 agonists, N-arachidonoyl aminophenol (AM 404) (KI: 2.57 μg/mL) [ ], Arvanil (KI: 1.28 μg/mL) [ ], and Olvanil (KI: 4.127 μg/mL) [ ], were purchased from Alomone Labs, Jerusalem BioPark (JBP) (Jerusalem, Israel) and utilized in a dose-dependent manner.

Techniques: Immunofluorescence, Expressing, Control, Fluorescence

Journal: Cells

Article Title: Functional Selectivity of Cannabinoid Type 1 G Protein-Coupled Receptor Agonists in Transactivating Glycosylated Receptors on Cancer Cells to Induce Epithelial–Mesenchymal Transition Metastatic Phenotype

doi: 10.3390/cells13060480

Figure Lengend Snippet: Immunofluorescence analysis of vimentin expression in response to CB1-agonists. CB1-agonists, AM-404, Arvanil, and Olvanil, significantly increased the expression of the epithelial–mesenchymal transition (EMT), vimentin, in SW-620 cells. Cells were fixed, permeabilized, blocked, and immunostained with primary mouse monoclonal IgG antibodies for vimentin, followed by goat anti-mouse AlexaFluor 488 (green) secondary antibodies. As an isotype control, we use normal mouse IgG. The fluorescence was calculated using Corel Photo-Paint, with the average of eight points subtracted by the background fluorescence and multiplied by the pixel density (20× objective). Statistical significance, as indicated by asterisks, was calculated with ANOVA and Fisher’s uncorrected LSD multiple comparisons post hoc test at a confidence level of 95%. ns = non-significant, **** p < 0.0001, *** p < 0.001.

Article Snippet: The CB1 agonists, N-arachidonoyl aminophenol (AM 404) (KI: 2.57 μg/mL) [ ], Arvanil (KI: 1.28 μg/mL) [ ], and Olvanil (KI: 4.127 μg/mL) [ ], were purchased from Alomone Labs, Jerusalem BioPark (JBP) (Jerusalem, Israel) and utilized in a dose-dependent manner.

Techniques: Immunofluorescence, Expressing, Control, Fluorescence

Journal: Cells

Article Title: Functional Selectivity of Cannabinoid Type 1 G Protein-Coupled Receptor Agonists in Transactivating Glycosylated Receptors on Cancer Cells to Induce Epithelial–Mesenchymal Transition Metastatic Phenotype

doi: 10.3390/cells13060480

Figure Lengend Snippet: The cells stably express a secreted embryonic alkaline phosphatase (SEAP) gene inducible by NF-kB and AP-1 transcription factors. Upon stimulation, RAW-Blue™ cells activate NF-kB and AP-1, leading to the secretion of SEAP, which is detectable and measurable when using QUANTI-Blue™, a SEAP detection medium (Invivogen). Quantitative spectrophotometry analysis of the effect of LPS and AM-404-, Arvanil-, and Olvanil-induced SEAP activity in the culture medium. The measurement of the relative SEAP activity was calculated as fold change in each compound (SEAP activity in medium from treated cells minus no cell background over SEAP activity in medium from untreated cells minus background). Results are the means of three separate experiments. Statistical significance, as indicated by asterisks, was calculated with ANOVA and Fisher’s uncorrected LSD multiple comparisons post hoc test at a confidence level of 95%.**** p < 0.0001, *** p < 0.001.

Article Snippet: The CB1 agonists, N-arachidonoyl aminophenol (AM 404) (KI: 2.57 μg/mL) [ ], Arvanil (KI: 1.28 μg/mL) [ ], and Olvanil (KI: 4.127 μg/mL) [ ], were purchased from Alomone Labs, Jerusalem BioPark (JBP) (Jerusalem, Israel) and utilized in a dose-dependent manner.

Techniques: Stable Transfection, Spectrophotometry, Activity Assay