Cannabidiol Suppresses Metastatic Castration-Resistant Prostate Cancer Progression and Recurrence through Modulating Tryptophan Catabolism

Abstract

Metastatic castration-resistant prostate cancer (mCRPC) is an aggressive phenotype of prostate cancer (PC). Tryptophan oxidative catabolism by indoleamine 2,3-dioxygenase-1 (IDO1) cleaves the indole ring to kynurenine (Kyn), an endogenous ligand for the aryl hydrocarbon receptor (AhR), which activates multiple tumorigenesis pathways. The IDO1-Kyn-AhR axis is aberrantly dysregulated in mCRPC. (-)-Cannabidiol (CBD) is a nonpsychoactive phytocannabinoid. CBD showed antitumor activities against human malignancies, including PC. CBD showed potent in vitro dose-dependent reduction of viability and clonogenicity of diverse human PC cell lines. CBD reduced the expression of IDO1 and AhR in PC cells. A daily 15 mg/kg oral dose of CBD for 30 days effectively suppressed the progression of the mCRPC CWR-R1ca-Luc cells xenografted in male nude mice. Continued CBD oral dosing for an additional 45 days suppressed the CWR-R1ca-Luc tumor locoregional and distant recurrences after the primary tumors' surgical excision. Collected CBD-treated tumors showed a reduced level of IDO1 expression. CBD-treated mice displayed a significant systemic reduction of Kyn. CBD is a novel, nonpsychoactive phytocannabinoid lead useful for the control of mCRPC via targeting the tryptophan catabolism.

Figure 1

Chemistry and binding mode of CBD at IDO1 crystal structure 6AZW. (A) Chemical structure of CBD. (B) CBD is extended at the substrate binding pocket and coordinated by SER 267 and PHE 214. (C) Space-filling representation of CBD showing the molecular binding architecture with the head (the cyclohexenyl ring), body (the phenyl ring), and tail (the n-pentyl side chain). (D) CBD phenolic moiety is held at the substrate binding site through contributing a H-bonding interaction with the SER 267 hydroxyl side chain and π–π stacking interaction with the PHE 214 phenyl side chain.

Figure 2

Effect of CBD treatments on PC cell line proliferation, clonogenicity, and migration using the MTT, colony formation, and wound-healing assays, respectively. (A) Dose–response line graph representing the percent PC cell proliferation with 0.2, 0.5, 1, 3, 6, and 9 μM CBD treatments over a 24 h culture period. (B) Dose–response line graph representing the percent PC cell clonogenicity at CBD 0.2, 0.5, 1, 3, and 6 μM treatments. (C) Photographs showing the CBD treatment effects on PC cell colony formation. (D) Comparison of the antiproliferative activities of CBD versus the standard IDO1 inhibitor 1-methyl-(−)-tryptophan (1MT) against the mCRPC cell line CWR-R1ca. (E) Micrographs showing the 0, 2, and 4 μM CBD treatments on CWR-R1ca cell migration over 16 h treatment periods. (F) Dose–response line graph translating the number of CWR-R1ca-migrated cells in the presence of 0, 2, and 4 μM CBD treatments over 16 h treatment course.

Figure 3

Overview of the IDO1 and AhR expression levels in PC cells. (A) Western blotting of IDO1 expression level in PC cell lines. (B) Densitometric quantification of IDO1 expression in PC cell lines. (C) Western blotting expression level assessment of AhR in PC cell lines. (D) Densitometric quantification of AhR in PC cell lines. (E) Expression of IDO1 and AhR in the nontumorigenic prostate epithelial cells RWPE-1. (F) Densitometric quantification of IDO1 and AhR in the RWPE-1 cells. Error bars indicate SD for each experimental group. “ns” indicates statistical nonsignificance. *Indicates statistical significance, p < 0.05 relative to the mCRPC CWR-R1ca cells.

Figure 4

Effect of CBD treatments on the expression levels of IDO1 and AhR in CWR-R1ca PC cells. (A) Immunoblots of IDO1 and AhR in CWR-R1ca cells treated with the vehicle control and 6 and 10 μM CBD over 16 h. (B) Densitometric analysis of IDO1 and AhR expression in CWR-R1ca cells. Scanning densitometry was obtained for all blots and carried out in duplicate, and the integrated optical density of each band was normalized to β-tubulin in the same blot. Bar graphs represent the mean relative protein expression percent. Error bars indicate SD, *p < 0.05, **p < 0.01, and ***p < 0.001, compared to their respective β-tubulin control group.

Figure 5

Effects of IDO1 knockdown on the mCRPC CWR-R1ca PC cell line clonogenicity and migration abilities. (A) Western blot validation of successful IDO1 knockdown in CWR-R1ca cells and comparison of the IDO1 expression in mock-type CWR-R1ca cells versus the IDO1-KD cells. (B) Densitometric comparison of the IDO1 expression in the mock-type CWR-R1ca cells versus the IDO1-KD cells. (C) Photographic imaging comparison of colony formation ability in the mock-type CWR-R1ca cells versus the IDO1-KD cells. (D) Bar graph comparative quantification of the percentage of clonogenicity of the mock-type CWR-R1ca cells versus the IDO1-KD cells. (E) Line graph showing timeline monitoring comparison of the average viability of seeded mock-type CWR-R1ca cells versus the IDO1-KD cells over a 72 h treatment period. (F) Microscopic imaging comparison of the migration ability of the mock-type CWR-R1ca cells versus the IDO1-KD cells. (G) Violin-plot comparative quantification of the migrated cell numbers of the mock-type CWR-R1ca cells versus the IDO1-KD cells. Error bars indicate SD for each experimental group. *Indicates statistical significance at p < 0.05.

Figure 6

Effect of daily CBD oral 15 mg/kg over 30 days on the mCRPC CWR-R1ca-Luc cell progression in nude mice. (A) Overview of the mCRPC progression model in an athymic nude mouse model. (B) Bioluminescence intensity monitoring of CWR-R1ca-Luc cells in the intact animal shows the oral effect of vehicle control and CBD 15 mg/kg daily oral treatments during the experiment course (30 days). (C) Bioluminescence comparison of animal organs for the VC- versus CBD-treated groups collected at the end of the study. (D) Collected mCRPC CWR-R1ca-Luc primary tumors of CBD- and VC-treated groups after the experiment completion. The top row shows the primary VC-treated group tumors, while the bottom row shows the tumors collected from the CBD-treated mice, 15 mg/kg, oral, 30 day dosing. (E) Bar graph comparing the average tumor weight in VC- versus CBD-treated groups at the progression study end. (F) Quantitative bioluminescence imaging represents the metastatic burden for the VC and CBD-treated groups at the study end. (G) Comparison of the level of Kyn in the VC- versus CBD-treated mice plasma samples at the conclusion of the progression study. (H) Timeline monitoring of the mean mice body weight over the experiment course from tumor cells xenografting until the day of sacrifice. (I) Line graph monitoring and comparing the average mean tumor volume in VC- versus CBD-treated groups over the 4-week dosing course. (E) Bar graph comparing the average tumor weight in VC- versus CBD-treated groups at the progression study end. (J) Comparison of Western blotting analysis of IDO1 expression in collected primary tumors of VC- versus CBD-treated groups in the progression model. (K) Densitometric quantification of the IDO1 expression in VC- versus CBD-treated progression model primary tumor lysates. *, **, and ***Indicate statistical significance for the CBD-treated versus VC-treated group at p < 0.01 and p < 0.001, respectively.

Figure 7

Suppressive effects of daily CBD oral 15 mg/kg treatments over 45 days on the locoregional and distant recurrences of the mCRPC CWR-R1ca-Luc cells in nude mice. (A) Overview of the animal study recurrence model. (B) Bioluminescence intensity imaging indicating CBD 15 mg/kg daily oral treatments prevented CWR-R1ca-Luc cells locoregional recurrence versus the VC-treated group, which showed 100% recurrence. (C) Visual bioluminescence imaging representation of animal organs for the VC-treated group versus CBD-treated group (D) collected at the study end. (E) Quantitative bioluminescence imaging assessment of the distant tumor recurrences burden for VC-treated versus CBD-treated groups at the study end. (F) Kynurenine plasma level comparison for VC-treated versus CBD-treated groups at the recurrence study end. (G) Line graph monitoring of the mean body weight for the VC-treated versus CBD-treated mice over the recurrence study model. **Indicates statistical significance for CBD-treated versus VC-treated group at p < 0.01.