Table 2 CBD in combination therapy for breast cancer

Synthetic cannabinoid receptor agonists inhibit tumor growth and metastasis of breast cancer

Preclinical (in vitro and in vivo)

In vitro: MDA-MB231, MDA-MB231-luc, MDA-MB468; In vivo: SCID mice, PyMT transgenic mice

Triple-Negative Breast Cancer (TNBC) (ER-, PR-, HER2-)

JWH-133 (CB2 agonist) and WIN-55,212-2 (CB1/CB2 agonist): 5 mg/kg/day i.p. in mice. In vitro concentrations not specified

Cell proliferation, migration, tumor growth, lung metastasis, apoptosis, COX-2/PGE2 signaling, c-Fos/c-Jun/Cdc42 activity, angiogenesis, Ki67 expression, CD31 expression

CB1 and CB2 receptors are overexpressed in primary human breast tumors. Synthetic agonists JWH-133 and WIN-55,212-2 inhibit cell proliferation and migration in vitro, reduce tumor growth and lung metastasis in vivo (40–50% reduction in tumor growth, 65–80% reduction in lung metastasis), delay mammary gland tumors in PyMT mice. Effects reversed by CB1/CB2 antagonists. They mediate tumor-suppressive effects via COX-2/PGE2 signaling and induction of apoptosis. They also modulate downstream molecules c-Fos, c-Jun, and Cdc42.

COX-2/PGE2 suppression, apoptotic pathways, c-Fos/c-Jun modulation

[23]

Cannabinoids reduce ErbB2-driven breast cancer progression through Akt inhibition

In vivo (MMTV-neu mice), Ex vivo (human tumor samples)

MMTV-neu mice; Human tumors (ErbB2-positive)

HER2-Positive Breast Cancer (ErbB2-positive)

THC: Peritumoral administration (Dosage not specified). JWH-133: Peritumoral administration (Dosage not specified).

Tumor growth, tumor number, lung metastases (incidence & severity), cell proliferation (Ki67), apoptosis (cleaved caspase 3), angiogenesis (CD31), MMP activity, Akt signaling

THC and JWH-133: Reduced tumor growth, tumor number, and lung metastases. Decreased cell proliferation, induced apoptosis, and impaired angiogenesis in tumors. Downregulated Akt signaling pathway. 91% of ErbB2-positive human breast tumors express CB2 receptors.

CB2 activation, Akt pathway inhibition, apoptosis induction

[24]

Bone cell-autonomous contribution of type 2 cannabinoid receptor to breast cancer-induced osteolysis

Preclinical (in vitro and in vivo)

Human MDA-MB-231, MCF7, Mouse 4T1 breast cancer cells, Calvarial bones of 2-day-old mice, Bone marrow macrophages

Triple-Negative Breast Cancer (TNBC) (ER-, PR-, HER2-) (MDA-MB-231), Hormone Receptor-Positive Breast Cancer (MCF7), Triple-Negative Breast Cancer (4T1)

CB2 agonists (e.g., HU308, JWH-133) administered in vitro and in vivo

Cell viability, osteoclastogenesis, osteolysis, osteoblast growth/differentiation, PI3K/AKT activity, Caspase-3 activity.

High micromolar concentrations of HU308 and JWH133 reduce the viability of breast cancer cells. Low nanomolar concentrations of HU308 and JWH133 enhance breast cancer cell-induced osteoclastogenesis and exacerbate osteolysis. HU308 and JWH133 induce PI3K/AKT activity in a CB2-dependent manner. CB2-selective activation and antagonism have potential efficacy in cancer-associated bone disease, but further studies are warranted. In the presence of conditioned medium from breast cancer cells, HU308 and JWH133 enhanced parathyroid hormone-induced osteoblast differentiation and the ability to support osteoclast formation. CB2 Activation enhances Breast Cancer-Induced Bone cell Activity and osteolysis via the PI3K/AKT pathway.

CB2 activation via PI3K/AKT pathway, bone metastasis modulation

[25]

Modulation of breast cancer cell viability by a cannabinoid receptor 2 agonist, JWH-015, is calcium dependent

Preclinical (in vitro and in vivo)

4T1, MCF7

Triple-Negative Breast Cancer (TNBC) (ER-, PR-, HER2-) (4T1), Hormone Receptor-Positive Breast Cancer (MCF7)

JWH-015, dose-dependent (e.g., A50 values: ~2.8 µM for 4T1, ~ 4.16 µM for MCF7)

Viability, apoptosis, metastasis, calcium flux, MAPK/ERK signaling

JWH-015 significantly reduced tumor viability and metastasis in vivo and induced apoptosis in vitro via a calcium-dependent mechanism. Effects were independent of CB1, GPR55, TRPV1/TRPA1 receptors, and Gαi signaling. MAPK/ERK modulation was implicated.

MAPK/ERK modulation, calcium-dependent apoptosis

[26]

Novel role of cannabinoid receptor 2 in inhibiting EGF/EGFR and IGF-I/IGF-IR pathways in breast cancer

Preclinical (in vitro and in vivo)

SUM159, MDA-MB231, MCF-7

ERα- (SUM159, MDA-MB-231), ERα+ (MCF-7)

JWH-015 (CNR2 specific agonist): In vitro - concentrations not specified; In vivo − 10 mg/Kg peri-tumoral injection for 4 weeks

Cell migration, cell invasion, NF-kB activation, EGFR/IGF-IR activation, tumor volume, tumor weight, STAT3/AKT/ERK activation, MMP secretion

CNR2 activation inhibits EGF/EGFR and IGF-I/IGF-IR signaling, reducing migration and invasion in both ERα- and ERα + breast cancer cells. In vivo, JWH-015 reduces tumor growth and inhibits EGFR/IGF-IR activation. Higher CNR2 expression correlates with better recurrence-free survival in breast cancer patients.

CNR2 activation inhibits EGF/EGFR and IGF-I/IGF-IR signaling

[27]

Appraising the “entourage effect”: Antitumor action of a pure cannabinoid versus a botanical drug preparation in preclinical models of breast cancer

Preclinical (in vitro and in vivo)

MCF-7 and T47D (ER+, PR+, HER2−); BT474 and HCC1954 (HER2+); MDA-MB-231 and SUM 159 (ER−, PR−, HER2−)

ER+/PR+, HER2+, Triple-Negative Breast Cancer (TNBC) (ER-, PR-, HER2-)

Pure THC and Botanical Drug Preparation (BDP) administered orally in mice (45 mg/kg THC)

Cell viability, tumor growth, interaction with standard therapies (e.g., tamoxifen, lapatinib, cisplatin)

BDP was more potent than pure THC in reducing cell viability and tumor growth. The “entourage effect” suggests that compounds in BDP enhance efficacy. No negative interactions with standard therapies were observed.

Entourage effect enhancing cannabinoid efficacy

[28]

Antitumor activity of abnormal cannabidiol and its analog O-1602 in taxol-resistant preclinical models of breast cancer

In vitro, In vivo

MDA-MB-231 and MCF-7

Triple-Negative Breast Cancer (TNBC) (ER-, PR-, HER2-)Triple-negative breast cancer (TNBC), ERα+ (MCF-7)

Abnormal cannabidiol (Abn-CBD) and O-1602 at varying concentrations, administered to cell cultures and animal models, 2 µM (in zebrafish xenograft model)

Cell viability, apoptosis induction, tumor growth inhibition, gene expression analysis

Abn-CBD and O-1602 significantly inhibited proliferation of Taxol-resistant breast cancer cells, induced apoptosis, and reduced tumor growth in xenograft models. They enhanced the efficacy of Taxol chemotherapy, suggesting potential to overcome drug resistance in breast cancer treatment. The compounds acted through non-CB1/CB2 cannabinoid receptors, highlighting a novel mechanism of action.

Non-CB1/CB2 mediated apoptosis and chemotherapy sensitization

[29]

Cannabinoid combination induces cytoplasmic vacuolation in MCF-7 breast cancer cells

Preclinical (in vitro)

MCF-7

Luminal A (ER+, PR+, HER2-)

C6 combination (THC, CBD, CBG, CBN); 40–60 µM total concentration

Cell cycle arrest, apoptosis, cytoplasmic vacuolation, lipid accumulation, lysosomal changes

The C6 combination induced cytoplasmic vacuolation through mechanisms involving autophagy and paraptosis. At lower doses (40 µM), it exerted cytostatic effects, while at higher doses (60 µM), it induced cytotoxicity. Markers of apoptosis and paraptosis were observed, including mitochondrial dilation and ER membrane involvement.

Autophagy and paraptosis induction

[30]

Combinatorial effects of cannabinoid receptor 1 and 2 agonists on characteristics and proteomic alteration in MDA-MB-231 breast cancer cells

Preclinical (in vitro)

MDA-MB-231

Triple-Negative Breast Cancer (TNBC) (ER-, PR-, HER2-)

CB1 and CB2 agonists in a 2:1 ratio (ACEA: GW405833)

Cell proliferation, invasion, lamellipodia formation, proteomic profile alteration

The 2:1 combination prominently inhibited colony formation, induced S-phase cell cycle arrest, and reduced invasion and lamellipodia formation. Proteomic analysis revealed alterations in pathways like ZPR1/SHC1/MAPK and AXL/VAV2/RAC1.

ZPR1/SHC1/MAPK & AXL/VAV2/RAC1 pathways

[31]

Cannabidiol enhances Atezolizumab efficacy by upregulating PD-L1 expression via the cGAS–STING pathway in triple-negative breast cancer cells

In vitro, in vivo

MDA-MB-231

Triple-Negative Breast Cancer (TNBC) (ER-, PR-, HER2-)

Cannabidiol (CBD), various administration methods

PD-L1 expression levels, cGAS-STING pathway activation, tumor cell apoptosis, synergistic effects of CBD and atezolizumab on immune response activation

CBD upregulates PD-L1 expression in TNBC cells via the cGAS-STING pathway, enhancing atezolizumab efficacy. Combination therapy induces stronger anti-tumor immune responses compared to atezolizumab alone

cGAS-STING immune activation, PD-L1 upregulation

[32]

Cannabidiol combination enhances photodynamic therapy effects on MCF-7 breast cancer cells

In vitro (cell lines)

MCF-7

Luminal A Breast Cancer (ER+, PR+, HER2-)

CBD: 1.25, 2.5, 5, 10, and 20 µg/mL (in vitro), Hypericin-Gold nanoparticles, PDT (594 nm, 5 J/cm2)

Cell morphology, LDH release, ATP levels, Trypan Blue exclusion, Immunofluorescence (Cytochrome c, Bcl-2, Bax, p53, PARP)

CBD induced cell death in MCF-7 cells in a dose-dependent manner (vacuolization, blebbing, floating). CBD + PDT combination therapy was effective in killing MCF-7 cells in vitro by induction of apoptosis (increased Cytochrome c, Bax, p53, PARP).

Cytochrome c, Bax, p53, PARP activation

[33]

In vitro evidence of selective pro-apoptotic action of the pure cannabidiol and cannabidiol-rich extract

Preclinical Study

MDA-MB-231 (breast cancer), PC-3 (prostate cancer), MCF-10 A (non-malignant breast cells), PNT2 (non-malignant prostate cells)

Triple-Negative Breast Cancer (TNBC) (ER-, PR-, HER2-) (MDA-MB-231)

0–15 µM pure CBD, CBD-rich Cannabis sativa extracts (extract B and extract D) containing equimolar CBD concentrations

Cell viability, morphological changes, endoplasmic reticulum stress-related apoptosis, gene expression involved in apoptosis and cell cycle control, ROS involvement

Both pure CBD and CBD-rich extracts decreased the viability of MDA-MB-231 and PC-3 cells in a concentration-dependent manner. Endoplasmic reticulum stress-related apoptosis and morphological changes were induced only in low-serum conditions. Non-malignant cell lines (MCF-10 A and PNT2) showed no alterations of viability, suggesting a selective action of CBD in tumor cells. Reactive oxygen species might be involved in the response mechanism. Significant changes in gene expression involved in apoptosis and cell cycle control were observed.

ER stress-related apoptosis, ROS generation

[34]

Rimonabant and Cannabidiol rewrite the interactions between breast cancer cells and tumor microenvironment

In vitro

MCF7, MDA-MB-231

MCF7 (ER

positive) and Triple-Negative Breast Cancer (TNBC) (ER-, PR-, HER2-)(MDA-MB-231)

Rimonabant and CBD; specific dosages not mentioned

Tumor-stroma interactions, growth factor secretion, tumor proliferation, angiogenesis, immune reprogramming

Both Rimonabant and CBD altered the release of factors involved in tumor proliferation, angiogenesis, and immune reprogramming, demonstrating anti-metastatic potential by reprogramming the tumor microenvironment

Tumor microenvironment modulation, anti-metastatic potential

[35]

The role of Cannabidiol and tetrahydrocannabivarin to overcome doxorubicin resistance in MDA-MB-231 xenografts in athymic nude mice

In vitro (2D & 3D cultures), In vivo (xenograft model), Transcriptomics, Proteomics

MDA-MB-231

Triple-Negative Breast Cancer (TNBC) (ER-, PR-, HER2-) (MDA-MB-231)

CBD: 2.5–30 µM (in vitro), 10 mg/kg (i.p. in vivo) THCV: 2.5–30 µM (in vitro), 15 mg/kg (i.p. in vivo) Doxorubicin (DOX): 2.5–30 µM (in vitro), 5 mg/kg (i.v. in vivo)

Cell viability (MTT assay in 2D & 3D cultures), Tumor volume (in vivo), RNA sequencing, Proteomics, Western blotting (histone modification markers)

CBD/THCV increased DOX cytotoxicity in DOX-resistant MDA-MB-231 cells (2D & 3D). Downregulated PD-L1, TGF-β, sp1, NLRP3, P38-MAPK, and upregulated AMPK, induced apoptosis. Inhibited H3k4 methylation and H2K5 acetylation.

PD-L1, TGF-β, P38-MAPK inhibition; AMPK upregulation

[36]

Anti-cancer effects of selective cannabinoid agonists in pancreatic and breast cancer cells

In vitro (cell lines)

MDA-MB-231 (TNBC), PANC1 (Pancreatic Cancer)

Triple-Negative Breast Cancer (TNBC) (ER-, PR-, HER2-) (MDA-MB-231)

L-759,633 (CB2 agonist), ACPA (CB1 agonist), ACEA (CB1 agonist): 1-250 µM for 72 h (MTS assay), 50 and 100 µM

Cell proliferation inhibition, clonogenicity suppression, apoptosis induction

CB1/CB2 agonists: Decreased cell proliferation and clonogenicity in both PANC1 and MDA-MB-231 cells. Upregulated pro-apoptotic Bax protein, downregulated anti-apoptotic Bcl-2 protein, induced apoptosis.

Bax upregulation, Bcl-2 downregulation, apoptotic pathways

[37]

Anticancer and chemosensitization effects of cannabidiol in 2D and 3D cultures of TNBC: Involvement of GADD45α, integrin-α5,-β5,-β1, and autophagy

Preclinical (in vitro and ex vivo)

MDA-MB-231, MDA-MB-468, MCF-10 A

Triple-Negative Breast Cancer (TNBC) (ER-, PR-, HER2-)T (MDA-MB-231 and MDA-MB-468), Immortalized Non-Tumorigenic Cells (MCF-10 A)

In vitro: CBD (1-2.5 µM for chemosensitivity, up to 10 µM for cytotoxicity), DOX (0.39 to 25 µM).

Cell viability, cell migration, gene expression (GADD45A, GADD45G, FASN, LOX, Integrins), protein expression (GADD45α, Integrins, Autophagy markers), cell cycle analysis, and organoid dissociation.

CBD showed higher IC50 values in 3D cultures compared to 2D cultures in TNBC cells. CBD alters the expression of GADD45A, GADD45G, FASN, LOX, and integrin genes (α5, β5) in MDA-MB-231 cells. CBD induces anti-migratory effects in TNBC cells by decreasing fibronectin, vimentin, and integrins α5, β5, and β1. CBD inhibits autophagy of TNBC cells by decreasing levels of Beclin1, ATG-5, ATG-7, and ATG-16. CBD pre-treatment increases the sensitivity of TNBC cells to doxorubicin (DOX). CBD induces cell cycle arrest at G0/G1 phase.

GADD45A, Integrins α5/β5 modulation, autophagy inhibition

[38]

Combination of cannabidiol with low-dose naltrexone increases the anticancer action of chemotherapy in vitro and in vivo

Preclinical (in vitro and in vivo)

A549 (human lung cancer), HCT116 (human colorectal cancer), MCF7 (human breast cancer)

ERα+ (MCF-7)

In vitro: CBD (1 µM), LDN (10 nM). NTX (10µM), GEM (~ IC20), OXP (~ IC20); In vivo: LDN (1.2 µg/mouse), CBD (35 µg/mouse), GEM (9 µg/mouse); All in a sequential treatment regimen

Cell number, viability, intracellular signaling protein expression (pAKT, AKT, pERK, ERK, CBR1, CBR2), tumor volume.

LDN before CBD is more effective than CBD before LDN at reducing cell numbers in vitro. LDN/CBD pre-treatment sensitizes cells to chemotherapy. LDN/CBD enhances the effect of gemcitabine in vivo. No significant toxicity was observed in mice treated with LDN/CBD. CBD, LDN and NTX had no significant effect on cell viability as single agents. Both CBR1 and CBR2 are present in all cell lines used. Combination treatments also led to decreased levels of pAKT and pERK in all cell lines.

CBR1/CBR2 modulation, AKT/ERK inhibition

[39]

Activation of cannabinoid receptors in breast cancer cells improves osteoblast viability in cancer-bone interaction model while reducing breast cancer cell survival and migration

Preclinical Study

MDA-MB-231, UMR-106

Triple-Negative Breast Cancer (TNBC) (ER-, PR-, HER2-)T

CB1 and CB2 agonists administered in vitro

Osteoblast viability, breast cancer cell survival, migration

CB receptor activation improved osteoblast viability while reducing breast cancer cell survival and migration; highlighted potential therapeutic benefits in cancer-bone interaction models

CB receptor activation modulates osteoblast-cancer interaction

[40]

Inhibition of cannabinoid receptor type 1 sensitizes triple-negative breast cancer cells to ferroptosis via regulating fatty acid metabolism

Preclinical (in vitro and in vivo)

MDA-MB-231, MDA-MB-436, HCC38, Hs578T, MCF-7, ZR75-1, T47-D, SKBR3, HEK 293 T, HCC1937, BT474, BT549, BT-20, HMEC

Triple-Negative Breast Cancer (TNBC) (ER-, PR-, HER2-), ERα+ (MCF-7)

Rimonabant (CB1 antagonist, concentrations not specified), erastin (ferroptosis inducer, concentrations not specified), RSL3 (ferroptosis inducer, concentrations not specified), LY294002 (PI3K inhibitor, concentrations not specified), PD98059 (MEK inhibitor, concentrations not specified).

Cell viability, lipid peroxide levels, malondialdehyde (MDA) levels, 4-hydroxynonenal (4-HNE) levels, cytosolic ROS production, intracellular glutathione (GSH) depletion, cell cycle analysis, tumor growth (in vivo), RNA sequencing, fatty acid analysis.

Inhibition of CB1 with rimonabant synergizes with erastin/RSL3 to inhibit TNBC cell growth in vitro and in vivo. This synergy is mediated by increased lipid peroxidation, MDA, 4-HNE, and ROS production, and GSH depletion. CB1 inhibition promotes G1 cell cycle arrest. CB1 regulates stearoyl-CoA desaturase 1 (SCD1)- and fatty acyl desaturase 2 (FADS2)-dependent fatty acid metabolism via PI3K-AKT and MAPK signaling pathways, thereby modulating ferroptosis sensitivity in TNBC cells. Dual targeting of CB1 and ferroptosis shows promise as a therapeutic strategy for TNBC.

PI3K-AKT & MAPK signaling, ferroptosis induction

[41]

Improved Therapeutic Efficacy of CBD with Good Tolerance in the Treatment of Breast Cancer through Nanoencapsulation and in Combination with 20(S)-Protopanaxadiol (PPD)

Preclinical (in vitro, in vivo)

4T1

Triple-Negative Breast Cancer (TNBC)

Nanoencapsulated CBD + PPD

Tumor inhibition rate, apoptosis, drug synergy, tolerance profile

Nanoencapsulation improved CBD’s therapeutic effects, achieving 82.2% tumor inhibition, while combination with PPD enhanced anticancer efficacy with good tolerance

Enhanced bioavailability, apoptosis induction

[42]

The Role of Cannabidiol and Tetrahydrocannabivarin to Overcome Doxorubicin Resistance in MDA-MB-231 Xenografts in Athymic Nude Mice

In vitro (2D & 3D cultures), In vivo (xenograft model)

Triple-Negative Breast Cancer (TNBC)

CBD: 2.5–30 µM (in vitro), 10 mg/kg (i.p. in vivo)

Chemosensitivity, apoptosis, immune modulation

CBD/THCV increased DOX cytotoxicity, downregulated immune checkpoint markers such as PD-L1 and TGF-β, overcoming drug resistance

The Role of Cannabidiol and Tetrahydrocannabivarin to Overcome Doxorubicin Resistance in MDA-MB-231 Xenografts in Athymic Nude Mice

PD-L1 & TGF-β downregulation, AMPK activation, histone modification

[36]