Pathways mediating the effects of cannabidiol on the reduction of breast cancer cell proliferation, invasion, and metastasis

Abstract

Invasion and metastasis of aggressive breast cancer cells are the final and fatal steps during cancer progression. Clinically, there are still limited therapeutic interventions for aggressive and metastatic breast cancers available. Therefore, effective, targeted, and non-toxic therapies are urgently required. Id-1, an inhibitor of basic helix-loop-helix transcription factors, has recently been shown to be a key regulator of the metastatic potential of breast and additional cancers. We previously reported that cannabidiol (CBD), a cannabinoid with a low toxicity profile, down-regulated Id-1 gene expression in aggressive human breast cancer cells in culture. Using cell proliferation and invasion assays, cell flow cytometry to examine cell cycle and the formation of reactive oxygen species, and Western analysis, we determined pathways leading to the down-regulation of Id-1 expression by CBD and consequently to the inhibition of the proliferative and invasive phenotype of human breast cancer cells. Then, using the mouse 4T1 mammary tumor cell line and the ranksum test, two different syngeneic models of tumor metastasis to the lungs were chosen to determine whether treatment with CBD would reduce metastasis in vivo. We show that CBD inhibits human breast cancer cell proliferation and invasion through differential modulation of the extracellular signal-regulated kinase (ERK) and reactive oxygen species (ROS) pathways, and that both pathways lead to down-regulation of Id-1 expression. Moreover, we demonstrate that CBD up-regulates the pro-differentiation factor, Id-2. Using immune competent mice, we then show that treatment with CBD significantly reduces primary tumor mass as well as the size and number of lung metastatic foci in two models of metastasis. Our data demonstrate the efficacy of CBD in pre-clinical models of breast cancer. The results have the potential to lead to the development of novel non-toxic compounds for the treatment of breast cancer metastasis, and the information gained from these experiments broaden our knowledge of both Id-1 and cannabinoid biology as it pertains to cancer progression.

Fig. 1

CBD up-regulates ERK phosphorylation and Id-2 expression. (A) Proteins from MDA-MB231 cells treated with 1.5 μM CBD (as previously described [21]) for 1 or 2 days were extracted and analyzed for Id-1, total ERK, pERK, or p38 by Western blot analysis. (B) Proteins from MDA-MB231 cells treated with CBD for 3 days were extracted and analyzed for Id-1, Id-2, or NFkappaB by Western blot analysis

Fig. 2

ERK partly mediates the inhibitory activity of CBD on cell growth and invasion. MDA-MB231 cells were treated for 3 days with vehicle (Control) or 1.5 μM CBD in the presence and absence of 0.1–0.5 μM U0126. Cell proliferation (A) and invasion (B) were measured using the MTT and Boyden chamber assays, respectively. Data are presented as relative proliferation or invasiveness of the cells, where the respective controls are set as 100%. (C) Proteins from MDA-MB231 cells treated with vehicle (control) or 1.5 μM of CBD for 3 days in the absence or presence of U0126 were extracted and analyzed for Id-1 by Western blot analysis. (*) indicates statistically significant difference from control (P < 0.05). (#) indicates statistically significant difference from CBD (P < 0.05)

Fig. 3

Production of ROS represents another factor involved in the inhibitory activity of CBD. MDA-MB231 cells were treated for 3 days with vehicle (Control) or 1.5 μM CBD in the presence and absence of 20 μM TOC. Cell proliferation (A) and invasion (B) were measured using the MTT and Boyden chamber assay, respectively. (C) Proteins from cells treated with vehicle (control) or 1.5 μM of CBD for 3 days in the absence or presence of TOC were extracted and analyzed for Id-1 by Western blot analysis. (D) The production of ROS was measured using 2′-7′Dichloro-dihydrofluorescein (Sigma-Aldrich). (*) indicates statistically significant differences from control (P < 0.05)

Fig. 4

CBD inhibits the expression of Id-1 and corresponding breast cancer proliferation and invasion in mouse 4T1 cells. (A) 4T1 cells were treated for 3 days with 1.5 μM CBD, proteins were extracted and analyzed for Id-1 expression. (B) 4T1 cells were collected and cell cycle analyzed using a desktop FACS Calibur with Cell Quest Pro software (BD Bioscience, CA). The distribution of cells in different cell cycle stages was determined according to their DNA content. (C) Invasion assays were carried out using the Boyden chamber assay. (*) indicates statistically significant differences from control (P < 0.05)

Fig. 5

CBD reduces primary tumor growth and metastasis of 4T1 cancer cells in an orthotopic mouse model. Primary tumors and subsequent secondary tumors (metastases) were generated in BALB/c mice by subcutaneous injection of 1 × 10⁵ 4T1 cells under the fourth major nipple. Treatment with CBD was initiated upon detection of the first palpable tumor (approximately 7 days). (A) The primary tumor volume was calculated by measuring the perpendicular largest diameters of the tumor with a caliper. (B) The weight of the tumors was also measured. (C) The visible lung metastases were measured using a dissecting microscope. (D) The average volume per metastatic foci was calculated as described in the methods. (*) indicates statistically significant differences from vehicle (P < 0.05)

Fig. 6

CBD reduces the number of metastatic foci in the syngeneic model of tail vein injection. Lung metastases were generated in BALB/c mice after tail vein injection of 5 × 10⁵ 4T1 cells. One day after the injection, the tumor-bearing mice were injected i.p. once a day with vehicle or 1 mg/kg CBD for 15 days. Visible lung metastases were counted and measured by using a dissecting microscope (A). Lung metastases measured included those (B) <1 mm, (C) 1–2 mm, and (D) >2 mm