Synthetic Cannabinoid Activity Against Colorectal Cancer Cells

Abstract

Introduction: Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide, and new therapeutic strategies are still required. Here we screened a synthetic cannabinoid library to identify compounds that uniformly reduce the viability of seven CRC cell lines. Material and Methods: Seven distinct CRC cell lines were treated with 10 μM cannabinoid compounds (from a library of 370 molecules) for 48 h, and cell viability was subsequently measured with MTS assay. Dose-response curves were conducted for compounds that were found to reproducibly reduce cell viability of one or more cell lines. Results: We identified 10 compounds from the library that were able to reduce cell viability of CRC cell lines (with an IC₅₀ ≤ 30 μM). Of these compounds, seven were specific for CRC cells, and six were effective in all CRC cell lines tested. Treatment with traditional phytocannabinoids (THC or CBD) was either ineffective or much less potent and only partially efficacious. Treatment with antagonists for the known cannabinoid receptors (alone or in combination) failed to block the activity of the most potent of identified compounds. Conclusion: We identified three families of cannabinoid compounds that reduce CRC cell viability through a noncanonical receptor mechanism. Future modification of these compounds may lead to the development of novel therapies to treat this disease.

Keywords: CBD; THC; colorectal cancer; synthetic cannabinoids.

FIG. 1.

Cannabinoid receptors are expressed in CRC cell lines. RT-qPCR was performed to measure the levels of mRNA for each cannabinoid receptor in the seven CRC cell lines, and the control HEK 293 and CCD 814 CoTr cells (A) CNR1, (B) GPR55, and (C) TRPV1. Data are relative to GAPDH mRNA levels and are normalized to the expression in SW480 cells. As noted in the text, CNR2 RT-qPCR failed to produce reliable amplification curves (due to low or absent mRNA levels). Error bars are SEM. In general, HT-29 and LS174 cells expressed significantly higher levels of CNR1 and GPR55 mRNA (p≤0.05). CRC, colorectal cancer; mRNA, messenger RNA; RT-qPCR, reverse transcribed-quantitative polymerase chain reaction; SEM, standard error of the mean.

FIG. 2.

Cannabinoid screening protocol. This schematic illustrates the procedure used for screening the 370 synthetic cannabinoid library against seven CRC cells.

FIG. 3.

Synthetic cannabinoid compounds reduce the viability of CRC cell lines. Cell viability was assessed with MTS assay 48 h after treatment with selected cannabinoid compounds at a concentration of 10 μM: (A) 5-epi CP 55,940, (B) HU-331, (C) PTI-1, (D) 3-epi CP 47,497 C-8 homolog, (E) CBD, and (F) THC. The DMSO control bar is included as a general reference as viability for each of the cell lines was normalized to its own vehicle (DMSO) treatment (conducted in triplicate). Error bars are SEM; *p≤0.05, **p≤0.01, ***p≤0.001.

FIG. 4.

Dose–response curves for selected cannabinoid compounds in CRC cell lines. Cell viability was assessed with MTS assay 48 h after treatment with selected cannabinoid compounds at concentrations of 100 nM, 333 nM, 1 μM, 3.3 μM, 5.6 μM, 10 μM, 33 μM, and 100 μM: (A) 5-epi CP 55,940, (B) HU-331, (C) (−) CP 47,497, and (D) CBD. Viability was normalized to vehicle (DMSO)-treated cells. Error bars are SEM.

FIG. 5.

Antagonists to canonical cannabinoid receptors do not prevent the reduction of cell viability. Cell viability was assessed in SW480 cells 48 h after treatment with 5-epi CP 55,940 at a concentration of 5 μM in the presence of vehicle, rimonabant (CB1 antagonist), SR 144528 (CB2 antagonist), ML-193 (GPR55 antagonist), SB-705498 (TRPV1 antagonist), or all four antagonists at 10 μM. Viability was normalized to vehicle (DMSO)-treated cells in four standard wells on each plate. Error bars are SEM. There were no statistically significant differences between 5-epi CP 55,940-treated groups, co-treated with either antagonist or vehicle. However, all of the 5-epi CP 55,940-treated groups displayed significantly reduced viability compared with their cognate vehicle controls (p≤0.05). SR 144528 and ML-193 modestly, but significantly, increased viability of SW480 cells (p≤0.05).

FIG. 6.

Structure of compounds that reduced viability of CRC cell lines below 50%. Shown are the structures of 10 synthetic cannabinoid compounds (compared with CBD and THC) that were shown to reproducibly reduce cell viability of CRC cells.