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Treatments combining CBD and PAC produce additive to synergistic inhibition of breast cancer cell viability. Cell viability was measured using the MTT assay. (A) 4T1 and MDA-MB231-luc-D3H2LN (LN 231) cells were treated for 2 days with vehicle, CBD or PAC. Specific dose ratios of CBD and Pac where then combined in (B) 4T1 and (C) MDA-MB231-luc-D3H2LN cells. Cell viability (%) was calculated as the MTT product absorbance in the treated cells/control cells × 100. These data were used to calculate (D) CI values as described in Methods. A CI value of <1, 1 and >1 indicates synergism, additivity and antagonism respectively (Chou et al., 1993). Data are the mean of at least three independent experiments; bars, ±SEM.
IC50 values were calculated using CompuSyn (Paramus, NJ, USA). To test for synergism, the combination index (CI) was also calculated using Compusyn where CI <1, = 1 and >1 indicates synergism, additive effect and antagonism, respectively, as previously described (Chou et al., 1993; Chou, 2006) and as previously published by our group (Marcu et al., 2010). Based on the classic isobologram for mutually exclusive effects relative to the end point of measurement, the CI value for x % inhibition is calculated as: CI = (D)1/(Dx)1 + (D)2/(Dx)2.
In the first set of experiments, mechanical allodynia was assessed in five groups of mice (n = 8 per group) using von Frey monofilaments of varying forces (0.07–4.0 g) applied to the mid-plantar surface of the right hind paw, with each application held in c-shape for 6 s using the up-down method of Dixon (1980). Mice were placed in individual Plexiglas compartments (Med Associates, St. Albans, VT, USA) on top of a wire grid floor suspended 20 cm above the laboratory bench top and acclimatized to the environment for 15 min before each test session. Baseline sensitivity to the monofilaments was assessed 1 day before the start of drug administration and continued weekly for 10 weeks. On experimental days 1, 3, 5 and 7, mice received the following two i.p. injections, spaced 15 min apart: group 1 – CRM vehicle, CRM vehicle; group 2 – CRM vehicle, 4.0 mg·kg −1 PAC; group 3 – CRM vehicle, 8.0 mg·kg −1 PAC; group 4 – 2.5 mg·kg −1 CBD, 8.0 mg·kg −1 PAC; 5.0 mg·kg −1 CBD, 8.0 mg·kg −1 PAC. Mechanical allodynia was not assessed on injection days. PAC and CBD doses were based on significant findings from Ward et al. (2011).
PAC-induced mechanical sensitivity was prevented by administration of CBD (2.5 – 10 mg·kg −1 ) in female C57Bl/6 mice. This effect was reversed by co-administration of the 5-HT1A antagonist WAY 100635, but not the CB1 antagonist SR141716 or the CB2 antagonist SR144528. CBD produced no conditioned rewarding effects and did not affect conditioned learning and memory. Also, CBD + PAC combinations produce additive to synergistic inhibition of breast cancer cell viability.
There was no effect of CBD on time spent in the white, CBD-paired compartment compared with CRM vehicle control, although there was a trend towards a decrease in the time spent in the CBD-paired compartment at the highest dose tested. One-way anova revealed no significant effect of treatment [F(3, 31) = 2.477, n.s.]. By comparison, morphine treatment significantly increased the time spent in the white, morphine-paired compartment compared with saline vehicle control [F(3, 30) = 15.66, P < 0.0001] (Figure 4 ). Furthermore, CBD treatment had no effect on the time to earn 10 reinforcers during the acquisition [F(3, 32) <1] or retention [F(3, 25) = 1.692, n.s.] sessions (Figure 5 ).
A novel strategy investigated in the present study is that of assessing the ability of CB-based pharmacotherapy to prevent the development of PAC-induced mechanical sensitivity as opposed to acutely reversing it. Other studies have demonstrated the ability of agents from other drug classes, including anticonvulsants (Xiao et al., 2007), antidepressants (Xiao et al., 2008) and opioids (Rahn et al., 2008), to reduce CIPN symptoms in rodents, but to date no one drug or drug class is considered to be effective for reversal of CIPN (Lynch et al., 2004). CIPN represents a neuropathic pain state with the unique possibility of aiming to prevent its onset with effective adjunctive treatment, as opposed to only attempting to reverse its symptoms following its onset. However, such investigations into prevention of PAC-induced CIPN in rodents are few. Interestingly, CBD has also recently been reported to protect against the onset of type I diabetic peripheral neuropathic pain (Toth et al., 2010), hepatic ischaemia/reperfusion injury (Mukhopadhyay et al., 2011), and retinal inflammation and degeneration (El-Remessy et al., 2008) in rodent models. While clinical trials are ongoing investigating the anti-inflammatory effects of CBD as a monotherapy in disease states such as inflammatory bowel disease and graft versus host disease, its efficacy at preventing the onset of neuropathic pain in humans remains to be determined.
Based on growing preclinical literature, the myriad of CBD’s pharmacological effects, from anti-neuropathic to anxiolytic and antipsychotic, may be mediated through either CB receptor-dependent and independent mechanisms or combinations thereof (Izzo et al., 2009). It is important from both a basic science mechanistic as well as drug discovery perspective to identify which of these are necessary and/or sufficient for CBD’s anti-neuropathic effects specifically. In the present study, we demonstrated that activation of 5-HT1A receptors is necessary for the protective effect of CBD against PAC-induced neuropathic pain, in that pretreatment with WAY100635 blocked this effect. CBD acts as a direct agonist at 5-HT1A receptors (Russo et al., 2005; Alves et al., 2010), and activation of the 5-HT1A receptor in the rostroventromedial medulla plays an important role in modulating the descending inhibitory pain pathway (Colpaert, 2006; Viisanen and Pertovaara, 2010). Importantly, 5-hydroxytryptaminergic drugs presently represent one of the only drug classes showing efficacy in the treatment of neuropathic pain in human clinical trials (Finnerup et al., 2010). 5-HT1A agonism has also been shown to be neuroprotective via attenuation of microglial activation and oxidative stress (Collier et al., 2011a,b2011b), two immune alterations relevant to CIPN. Results from the present study failed to show a role for CB1 or CB2 receptor activation in CBD’s anti-neuropathic effect. Although CBD has no appreciable affinity for CB1 or CB2 receptors, some evidence suggests that it can act as an indirect CB agonist via enhancement of eCB levels (Bisogno et al., 2001; Campos et al., 2013). However, our results are in agreement with the previous report by Comelli et al. (2008) demonstrating that CBD’s anti-hyperalgesic effect did not involve CB1 and CB2 receptors. Others have shown that neither CB1 nor CB2 receptor activation was involved in CBD’s neuroprotective (Sagredo et al., 2007; 2011 , ) or anti-inflammatory (Costa et al., 2004) effects in other rodent models, whereas CBD-induced tail flick analgesia was blocked by co-administration of the CB1 antagonist SR141716 (Maione et al., 2011). CB1 receptor involvement in the pharmacological effects of CBD on non-nociceptive behaviours has also been reported (Casarotto et al., 2010; Do Monte et al., 2013). Additionally, CBD binds with moderate affinity to TRPV1 (vanilloid) receptors and important nociceptive modulators, and anti-neuropathic effects of CBD have been shown to depend upon TRPV1 activation (Comelli et al., 2008), while acute antinociceptive effects have not (Maione et al., 2011). Taken together with these other findings, our results suggest that specific pharmacological effects of CBD, such as its activity at 5-HT1A and TRPV1 receptors, mediate CBD’s anti-neuropathic effects, while its activity at other targets, including CB receptors, may be more important for other actions.
The mouse and human breast cancer cell lines used were 4T1 (obtained from ATCC) and MDA-MB231-luc-D3H2LN (obtained from Caliper; Jenkins et al., 2005) cells respectively. Cell lines were maintained at 37°C and 5% CO2. In all experiments, the different cell populations were first cultured in RPMI media containing 10% FBS. Cells were then seeded into 96-well plates in 10% FBS and on the first day of treatment the media was replaced with vehicle control or drug in RPMI and 0.1% FBS as previously reported (McAllister et al., 2005). The media with the appropriate compounds were replaced every 24 h.
Effect of WAY100635 pretreatment (1.0 mg·kg −1 , i.p.) on CBD prevention of PAC-induced mechanical allodynia in female C57Bl/6 mice. Baseline sensitivity to von Frey filaments was assessed on the day before drug administration and continued weekly for 10 weeks. Mice received the following three i.p. injections spaced 15 min apart on days 1, 3, 5 and 7: saline, CRM vehicle, CRM vehicle; saline, CRM vehicle, 8.0 mg·kg −1 PAC; saline, 5.0 mg·kg −1 CBD, 8.0 mg·kg −1 PAC; 1.0 mg·kg −1 WAY100635, 5.0 mg·kg −1 CBD, 8.0 mg·kg −1 PAC. Two-way anova revealed significant effects of treatment [F(3, 280) = 24.66, P < 0.0001] and time [F(9, 280) = 5.058, P < 0.001] and no significant interaction (F <1.0). Bonferroni post-test revealed a significant increase in the sensitivity of the PAC group and the WAY/CBD/PAC groups compared with Veh/Veh/Veh. In contrast, the Veh/CBD/PAC group did not differ significantly from the Veh/Veh/Veh group on mechanical sensitivity. X-axis: time points pre- or post-day first injection. Y-axis: threshold pressure to elicit hind paw withdrawal from von Frey filament. Data points represent the mean and SEM, n = 8 per group.