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. 2012 Nov 15;214(3):314-9.
doi: 10.1016/j.toxlet.2012.08.029. Epub 2012 Sep 8.

Cannabidiolic Acid, a Major Cannabinoid in Fiber-Type Cannabis, Is an Inhibitor of MDA-MB-231 Breast Cancer Cell Migration

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Cannabidiolic Acid, a Major Cannabinoid in Fiber-Type Cannabis, Is an Inhibitor of MDA-MB-231 Breast Cancer Cell Migration

Shuso Takeda et al. Toxicol Lett. .
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Abstract

Cannabidiol (CBD), a major non-psychotropic constituent of fiber-type cannabis plant, has been reported to possess diverse biological activities, including anti-proliferative effect on cancer cells. Although CBD is obtained from non-enzymatic decarboxylation of its parent molecule, cannabidiolic acid (CBDA), few studies have investigated whether CBDA itself is biologically active. Results of the current investigation revealed that CBDA inhibits migration of the highly invasive MDA-MB-231 human breast cancer cells, apparently through a mechanism involving inhibition of cAMP-dependent protein kinase A, coupled with an activation of the small GTPase, RhoA. It is established that activation of the RhoA signaling pathway leads to inhibition of the mobility of various cancer cells, including MDA-MB-231 cells. The data presented in this report suggest for the first time that as an active component in the cannabis plant, CBDA offers potential therapeutic modality in the abrogation of cancer cell migration, including aggressive breast cancers.

Conflict of interest statement

Conflict of interest statement

The authors declare that there are no conflicts of interest in this study

Figures

Fig. 1
Fig. 1
Chemical structures of CBDA and CBD. In the fiber-type cannabis plant, the concentration of CBD is much lower than that of its precursor CBDA. CBD is formed artificially from CBDA by non-enzymatic decarboxylation during extraction step (Yamauchi et al., 1967).
Fig. 2
Fig. 2
Effect of CBDA on the vertical migration of highly aggressive human breast cancer MDA-MB-231 cells. (A) Morphologies of two human breast cancer cell lines; MCF-7 cells (a) and MDA-MB-231 cells (b). MCF-7 cells display epithelial morphology (collective) and MDA-MB-231 cells display single elongated morphology (mesenchymal). (B) MDA-MB-231 cells were exposed for 12 h to CBDA (5, 10, 25 μM) and CBD (5, 10, 25 μM). After the treatments, cell viability was measured according to the methods described in Section 2. Data are expressed as the percent of vehicle-treated group (indicated as Cont.), as mean ± S.D. (n = 6). *Significantly different (p < 0.05) from the vehicle-treated control. (C) Transwell migration assays (vertical migration) were performed to determine MDA-MB-231 cell migration 12 h after treatments with 5 μM, 10 μM, or 25 μM CBDA and 5 μM CBD. Data are expressed as the percent of vehicle-treated group (indicated as Cont.), as mean ± S.D. (n = 8). *Significantly different (p < 0.05) from the vehicle-treated control. N.D., not detectable due to complete inhibition of the migration.
Fig. 3
Fig. 3
Effect of CBDA on the horizontal migration of MDA-MB-231 cells. (A) Wound-healing assays (horizontal migration) were performed to determine the effects of CBDA or CBD on MDA-MB-231 cell migration. (a) Representative images of the migrating cells were captured 12 h after vehicle (indicated as Cont.), 5 μM or 25 μM CBDA and 5 μM CBD treatments. (b) Migration data presented in panel (a) was assessed on the basis of percent wounded area filled in. Data are expressed as the percent of vehicle-treated group (indicated as Cont.), as mean ± S.D. (n = 8). (B) Migration data was assessed on the basis of percent wounded area filled in 12, 24, or 48 h after treatments with 25 μM CBDA. Data are expressed as the percent of vehicle-treated group (indicated as Cont.), as mean ± S.D. (n = 8) *Significantly different (p < 0.05) from the respective vehicle-treated controls.
Fig. 4
Fig. 4
COX-2 inhibitory activity of CBDA is not essential to attenuate MDA-MB-231 cell migration. Transwell migration assays were performed to determine the effects of COX-2 selective inhibitors (25 μM CBDA or 25 DuP-697) and COX-1selective inhibitor (25 μM SC-560) on MDA-MB-231 cell migration 12 h after their respective treatments. Data are expressed as the percent of vehicle-treated group (indicated as Cont.), as mean ± S.D. (n = 8). *Significantly different (p < 0.05) from the vehicle-treated control. N.D., not detectable due to complete inhibition of the migration.
Fig. 5
Fig. 5
CBDA stimulates RhoA activity. (A) RhoA affinity pull-down assays were used to determine the level of active RhoA according to the methods described in Section 2. Resulted pull-down samples were subjected to Western blot analyses using an anti-RhoA antibody. Active RhoA was increased by 25 μM CBDA (indicated as +) in a time-dependent manner. Western blot analyses were also performed using an anti-RhoA antibody specific to RhoA phosphorylated at Ser188 and a β-actin antibody. (B) Results are expressed as the ratio of active RhoA to total RhoA protein in each cell lysate. Data are expressed as the fold change vs. vehicle-treated group (indicated as −), as mean ± S.D. (n = 3). *Significantly different (p < 0.05) from the vehicle-treated control.
Fig. 6
Fig. 6
CBDA inhibits PKA activity. (A) (a) A representative photograph of phosphorylated bands and non-phosphorylated bands of PKA-specific substrate peptide in MDA-MB-231 cells incubated with vehicle (time 0, lane 3), vehicle (48 h, lane 4), and 25 μM CBDA (48 h, lane 5) was shown. For positive and negative controls, the reactions were performed in the presence (lane 1) or absence (lanes 2) of PKA catalytic subunit, respectively. An arrow in the image indicates wells that samples are applied. (b) The relative intensity of phosphorylated bands in MDA-MB-231 cells is shown. Data are expressed as fold change vs. non-CBDA-treated group (left panel; lanes 4 vs. 5), as mean ± S.D. (n = 3). *Significantly different (p < 0.05) from the vehicle-treated control. (B) MDA-MB-231 cells were treated with vehicle (indicated as Cont.) or 25 μM CBDA for 48, 72, or 96 h. After the treatments, cell viability was measured according to the methods described in Section 2. Data are expressed as the percent of vehicle-treated group, as mean ± S.D. (n = 6). *Significantly different (p < 0.05) from the vehicle-treated control. N.S., not significant. (C) A model of CBDA-mediated RhoA activation through PKA inhibition.

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