History and Purpose The psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC) and the non-psychoactive cannabinoid cannabidiol (CBD) can both reduce cancer progression each through distinct anti-tumour pathways. (CBD). THC activates CB1 and CB2 receptors and is the primary psychoactive cannabinoid in CS as a result of its interactions with CB1 receptors in the CNS (Pertwee 2006 CBD is the second most abundant cannabinoid in CS but does not interact efficiently with CB1 and CB2 receptors and is not psychoactive (Showalter synthesis of ceramide leading to endoplasmic reticulum stress and autophagy-mediated cell death (Carracedo and in addition discovered that CBD was also effective at inhibiting GBR 12783 dihydrochloride advanced stages of metastasis. Based on these results we then screened compounds and discovered a cannabinoid analogue that was more active than CBD at down-regulating Id1 and was also a CB2 selective agonist that could target CB2 receptor anti-tumour pathways. We present mechanistic data unique to this analogue that demonstrates inhibition of advanced stages of metastasis in preclinical models leading to prolonged survival. Methods Cell culture and drugs All cell lines were cultured as we previously described (McAllister experiments ethanol stocks of CBD and O-1663 were dissolved in a solution containing 2% ethanol 2 Tween 80 and 96% saline. Mouse models of breast cancer For the studies 6 week old female mice were used. Ten mice per group were used for the orthotopic studies and 6-8 mice per group were used for the i.v. model of metastasis. Mice were cared for as we previously described (McAllister analyses Rabbit Polyclonal to WAVE1. were conducted when appropriate. Survival between groups was compared using a log-rank Mantel-Cox test. < 0.006) (Supporting Information Fig. S1A-C). The potency of CBD at targeting metastasis in the i.v. model was similar to that previously reported by our group using the 4T1 orthotopic model of metastatic progression (McAllister is necessary for the anti-metastatic activity of GBR 12783 dihydrochloride CBD. (A) Immunohistochemical detection of Id1 and Ki67 was performed in lung tissues of vehicle (left) and CBD (right) treated 4T1-derived tumours. Nuclei are visible ... To further confirm the correlation between the effects of CBD and inhibition of Id1 expression in culture and vivo in human breast cancer cells we established stable pooled populations of MDA-MB231 cells expressing Id1shRNA (Supporting Information Fig. S3A). We observed similar reductions in cell proliferation and invasion rate in the culture and metastasis in MDA-MB231 cells expressing Id1shRNA or in parental MDA-MB231 cells GBR 12783 dihydrochloride treated with CBD (Supporting Information Fig. S3B-D). CBD produces a dose-dependent inhibition of metastasis in advanced stages of breast cancer progression CBD was most effective in targeting metastatic foci ≥2 mm suggesting that the compound could be effective at inhibiting the growth of secondary tumours even after their initial establishment in lungs. We therefore treated mice at a time point where visual lung metastatic foci were already formed (day 7 Figure ?Figure2A).2A). We found that CBD dose-dependently reduced the growth of established lung metastatic foci reduced the formation of new metastatic foci and increased survival (Figure 2B-D). While the median increase in survival was only a day a subset of animals did live 3-5 days longer (< 0.02). Based upon these findings we expected that synthesis of more GBR 12783 dihydrochloride active analogues based upon CBD would result GBR 12783 dihydrochloride in the development of a compound that could produce more robust inhibition of advanced stages of metastasis. Figure 2 CBD reduces the formation of metastatic foci and increases survival in advanced stages of metastatic progression. Lung metastases were generated in BALB/c mice after i.v. injection of 2 × 104 mouse 4T1 cells. (A) The pictures are representative … O-1663 is more active than CBD at inhibiting cell proliferation invasion and Id1 expression Our past studies (McAllister < 0.1) whereas O-1663 produced a medium increase in survival of 30 days (< 0.006). In the group treated with O-1663 50 of the mice were still alive and demonstrated no signs of disease progression GBR 12783 dihydrochloride at time of killing (2 months). Importantly few visible lung metastatic foci were present in 20% of these mice (Supporting Information Fig. S6A). Figure 6 O-1663 produces a significant inhibition of advanced stage breast metastasis. Lung metastases were generated in BALB/c mice by i.v. injection of 2 × 104 mouse 4T1 or 0.25 × 106 human MDA-MB231-luc-D3H2LN cells. (A B) One week after the.