"We really don't have a lot of evidence in dogs at this time to say, is this going to work? And in these different ideas of treatment in cancer or other aspects," Hocker said.
Sam Hocker hopes research will pave the way to a possible alternative to treat cancer in dogs
Guelph researchers hope to find out whether cannabis can be used as a possible treatment for bladder cancer in dogs as part of one of the first studies exploring the subject since legalization.
Increase to harm reduction
"We're providing harm reduction by talking about it," she said.
All the studies carried out on patient’s specimens were approved by the Institutional Ethical Committee (Ospedale San Raffaele, Milan, protocol URBBAN, Rev. February 2nd, 2014, approval date March 3rd, 2014) and the specific informed consent was obtained pre-emptively. We also declare that all the experiments involving animals were approved by the Institutional Animal Care and Use Committee (Ospedale San Raffaele, Milan, IACUC n. 631) and the Italian Ministry of Health (Office for Animal Health and Veterinarian Drugs). All the experimental procedures involving human or animal biologic material were carried out in compliance with the approved guidelines.
A sample size of 19 cases was calculated from an expected effect size “d” of 0.7 (expression levels of normal vs tumour or CB1 vs CB2) tested with a paired 2-tailed T-test with a power of 0.80 and an α probability of 0.05 (G*Power Software). Statistical analysis was performed using GraphPad Prism Software, (San Diego, USA).
In conclusion, our data associate the well-known anti-metastatic properties of cannabinoids 19 ,44 ,45 , with a molecular mechanism involving CB2 that induces SL metabolism changes. This finding and the selective tumour cytotoxicity may warrant further studies to support the use of non psycho-tropic CB2 agonists as novel adjuvant treatment in transitional BC, aimed at preventing both tumour recurrence and progression.
Lipid extraction and determination
In vitro, the apoptotic phenotype of cells (annexin-V + /PI + ) increased 6.7 ± 0.3 fold (p < 0.01) 48 hrs post JWH015 (Suppl. Fig. S5A). Treated cells also accumulated in the sub-G1 phase (28.2 ± 1 vs 2.2 ± 0.9% in control cells, p < 0.01) (Suppl. Fig. S5B). Apoptosis was confirmed by activated form of caspase 3 that increased following JWH015 ( Fig. 3B ). Caspase induction was not detectable earlier than 24 hrs after CB2 agonist exposure on RT112 cells, even in the presence of rapamycin ( Fig. 3C ). After an initial up regulation detected 24–48 hrs post drug exposure, AKT phosphorylation (pAKT) was significantly reduced at day 3 and 4, consistently with the cytotoxic effect ( Fig. 3D ). The up regulation of pAKT in RT112 cells 24 hrs post JWH015 was reversed by co-treatment with CB2 antagonist (SR144528) but not with CB1 antagonist (rimonabant) ( Fig. 3E ), suggesting a selective receptor-dependent effect.
Furthermore, the inactive form of c-SRC, which is typically phosphorylated in Tyr-527, was up-regulated over time in RT112 treated with the CB2 agonist ( Fig. 4D ). Finally, the effect of JWH015 on cell migration was partly reverted by the AMP-dNM-mediated inhibition of SL metabolism ( Fig. 4E,F ) and CB2 genetic silencing completely prevented the JWH015-mediated increase of inactive pSRC (not shown).
Plasma membrane-associated enzymatic activities of β-galactosidase, β-glucosidase, GBA1 and, GBA2, β-hexosaminidase, and α-galactosidase were determined in living cells plated in 96-well microplates at a density of 3.3 × 10 4 cells/cm 2 by using specific fluorogenic substrates as previously described 49 . The artificial substrates were solubilized in DMEM-F12 medium (Invitrogen) without phenol red or serum at different final concentrations (see Suppl. Methods for details). At various times, aliquots of the medium were analyzed fluorometrically by using a microplate reader (Victor, Perkin Elmer) with the addition of 20 volumes of 0.25 M glycine (Sigma), pH 10.7. The data were expressed as n/pmoles of converted substrate/mg cells proteins/hr. Control experiments were performed to exclude any activity released from lysosomes and/or from other intracellular sites. The enzymatic activities associated with total cell lysates were determined by using fluorogenic substrates as previously described 17 . Sphingosine kinase 1 (SK1) activity was assayed as previously described 50 . For the analysis of S1P production, cells were pulsed with [1- 3 H]sphingosine for 45 min as previously described 51 .
Thus, the effects CB2-receptor activators on BC survival and motility and their interactions with SL biochemical pathways represent an interesting aspect still poorly characterized.
Consistently with previous data 19 ,20 , we found that, in primary BC, the expression of CB2 in the neoplastic lesion is higher when compared to the paired normal urothelial and stromal counterparts. Our results also show that, like in other tumours 21 ,22 ,23 CB2 receptors may selectively control some cellular functions in BC cells.