In this study, the phenotypic effects of CBD were assessed using androgen-sensitive (LNCaP) and androgen-insensitive (DU145, PC-3) prostate cancer cell lines. The MTT assay was used to measure cell viability following 72 h treatment with various doses of CBD (0–100 μM). Under serum deprivation conditions, CBD significantly reduced the viability of all three cell lines, with IC₅₀ values of 1.5 μM (DU145), 2.9 μM (PC-3), and 2.6 μM (LNCaP) (Figure 1A). Because these cell lines are routinely grown in media containing serum, we also tested the effect of CBD in the presence of serum, which has previously been shown to reduce the efficacy of cannabinoids in prostate cells.⁸ As expected, IC₅₀ values were higher in cells grown with serum, specifically, 12.3 μM (DU145), 10.5 μM (PC-3), and 18.0 μM (LNCaP) (Figure 1B). Notably, under these conditions, the androgen-independent DU145 and PC-3 cell lines were more sensitive to CBD treatment than the androgen-dependent LNCaP cells. To further investigate the inhibition of DU145 and PC-3 cell viability by CBD, total cell numbers were assessed using flow cytometry. Treatment with an IC₅₀ dose of CBD significantly reduced cell counts for both cell lines, indicating a possible inhibitory effect on cell proliferation (Figure 1C). Additionally, clonogenic assay analysis revealed that 48 h CBD pretreatment significantly reduced the number of PC-3 cell colonies formed after 7 days by approximately 25% (p = 0.03) (Figure 1D–F), indicating that CBD reduces the ability of the cells to survive and proliferate following treatment.

To better understand how CBD reduces the viability of prostate cells, fluorescence microscopy was used to assess the effects of various CBD doses on cell proliferation and apoptosis in DU145 and PC-3 cells. Cells were treated with CBD (1, 5, or 10 μM) in the presence of serum and assessed using high-content screening microscopy. After 72 h treatment, CBD significantly reduced DU145 cell confluency at doses of 5 μM (p = 0.02) or 10 μM (p < 0.001) and reduced PC-3 cell confluency at a 10 μM dose (p < 0.001) (Figure 1G), indicating inhibition of cell proliferation. CBD did not reduce nucleus area or circularity, both indicative of cell death, at either time point at any of the doses tested (Supplementary Figure S1). Similarly, CBD did not significantly increase YO-PRO or propidium iodide positivity or reduce the fraction of healthy cells in either cell line (Figure 1H, Figure S2). Representative images of CBD-treated cells are shown (Figure 1I,J). The above findings were supported by flow cytometry analysis indicating that 48 h CBD treatment induced no significant pro-apoptotic effect in DU145 or PC-3 cells (Supplementary Figure S3). In agreement with the current study, a previous study reported that treatment with CBD in the presence of serum did not increase caspase activity or TUNEL positivity in DU145 or PC-3 cells.⁸ The above findings suggest that in cells grown with serum CBD reduces prostate cancer cell viability primarily through inhibition of cell proliferation, rather than through induction of apoptosis.

One of the major challenges in elucidating cannabinoid mechanisms of action is identifying the receptor targets that mediate cannabinoid phenotypic effects. Cannabinoids play roles in many physiological processes and can modulate the activity of a wide range of receptors and targets.²⁷ Common targets of cannabinoids in cancer cells include the major cannabinoid receptors, CB₁ and CB₂, the transient receptor potential vanilloid 1 (TRPV1) ion channel, and the putative novel cannabinoid receptor GPR55.^15,22,28,29 All the above targets have previously been demonstrated to be present in prostate cancer cells.^8,30−32 To identify the receptor targets of CBD, prostate cancer cells were pretreated with selective antagonists/agonists of common cannabinoid receptor targets, before assessing the effects of CBD on cell viability. Specifically, cells were pretreated with SR141716 (CB₁ antagonist), SR144528 (CB₂ antagonist), capsazepine (TRPV1 channel antagonist), or lysophosphatidylinositol (LPI) (GPR55 agonist), before 72 h treatment with an IC₅₀ dose of CBD. However, no significant difference in viability was observed in cells pretreated with the any of the antagonists/agonists compared to cells treated with CBD alone, in any of the cell lines (Figure 2). These results suggest that the CBD-induced reduction in prostate cancer cell viability does not require the activation of CB₁, CB₂, or TRPV1 or the antagonism of GPR55. In agreement with these findings, Mahmoud et al. recently reported that the effect of CBD on cell viability was not blocked by antagonists of CB₁, CB₂, or TRPV1 in the TRAMP cell line model of prostate cancer.¹² However, cannabinoids can modulate the activity of cannabinoid receptors through mechanisms other than direct activation or antagonism. For example, CBD can act as a negative allosteric modulator of CB₁, altering receptor conformation and ligand-binding activity.^33,34 Some studies report that CBD acts as an inverse agonist at CB₂, while others identify CBD as a partial CB₂ agonist.^34,35 Knocking down these targets using siRNA could provide insight into whether CBD is indirectly modulating the activity of these receptors. Alternatively, CBD may reduce the viability of prostate cancer cells primarily by interacting with many other cannabinoid targets. Previous studies have shown that cannabinoids can alter cancer-related processes through interactions with the transcription factor peroxisome proliferator activated receptor gamma (PPARγ), the mitochondrial protein voltage-dependent anion-selective channel 1 (VDAC1), the ion channels TRPM8 and transient receptor potential ankyrin 1 (TRPA1), serotonin receptors, and steroid receptors, among many other targets.^12,36−39

While cannabinoid mechanisms have been extensively studied in certain cancer types, for example, glioblastoma and breast cancer, the mechanisms driving the phenotypic effects of cannabinoids in prostate cancer are not fully understood. Increased cell proliferation in cancer is associated with increased activity of cyclins and CDKs, which drive cell cycle progression.⁴⁰ Inhibition of cell cycle progression is a useful therapeutic strategy in cancer, and several CDK inhibitors have been approved for cancer treatment.⁴¹ Some existing studies show that cannabinoids can reduce the levels of expression of cyclins and CDKs in cancer cells. In multiple myeloma and pancreatic cancer, for example, inhibition of cell proliferation by CBD was accompanied by reduced expression of cyclin D.^15,18 In gastric cancer, CBD inhibited the formation of the CDK2/cyclin E complex, which drives cell cycle progression.⁵ Having shown that CBD inhibits prostate cancer cell proliferation, we investigated whether CBD treatment modulates the expression of cyclins and CDKs in these cells. To determine whether CBD induces G1/S phase cell cycle arrest in prostate cancer cells, we measured the expression of CDK2, CDK4, and cyclin D3, which promote the G1/S phase cell cycle transition. In DU145 cells, CBD significantly reduced the expression of CDK2 (p = 0.049) and CDK4 (p = 0.04) (Figure 3A). Additionally, CBD reduced the level of cyclin D3 expression by approximately 20%, though this was not statistically significant. Furthermore, CBD significantly reduced the expression of cyclin D3 (p = 0.0002) and CDK2 (p = 0.04) in PC-3 cells (Figure 3A). These results indicate that CBD may induce G1/S phase cell cycle arrest in prostate cancer cells by reducing the level of expression of key proteins that drive the G1/S phase transition. Our results complement previous findings that CBD increases the expression of the CDK inhibitors p21 and p27^kip in prostate cancer cells.⁸

To assess whether CBD induces cell cycle arrest at the G2/M checkpoint, we also measured the expression of CDK1, a key regulator of cell cycle progression primarily involved in the G2/M phase transition, and CDK7, which also drives G2/M phase progression. CBD significantly reduced CDK1 expression in both DU145 (p < 0.0001) and PC-3 (p = 0.02) cells, with a particularly strong effect in DU145 cells (Figure 3A). CBD had no significant effect on CDK7 expression in either cell line (Supplementary Figure S4). These results provide evidence that CBD induces G2/M cell cycle arrest through downregulation of CDK1. Some evidence suggests that the psychoactive plant-derived cannabinoid THC inhibits breast cancer cell proliferation by reducing the expression of CDK1.⁴² Additionally, one recent study showed that a synthetic CBD analogue inhibited CDK1 mRNA expression in a model of cardiac fibrosis.⁴³ However, to the best of our knowledge, this study provides the first evidence that CBD reduces CDK1 expression in cancer. Together, the above results indicate that CBD downregulates key proteins that drive cell cycle progression through both the G1/S and G2/M checkpoints, further supporting our findings that CBD inhibits prostate cancer cell proliferation.

Phosphorylation and activation of the protein kinase AKT promotes cancer cell proliferation, survival, and invasiveness.⁴⁴⁻⁴⁶ AKT hyperphosphorylation is a common feature of prostate cancer, with increased AKT activity observed in 50% of prostate cancers.⁴⁷ Previous studies show that CBD reduces AKT phosphorylation in glioma, multiple myeloma, leukemia, and breast cancer, accompanied by reduced cell proliferation, migration, and invasion, and increased apoptosis.^{3,13,18,48,49} Here, we used Western blotting to assess the effect of CBD treatment on AKT signaling. CBD had no significant effect on AKT phosphorylation in PC-3 cells (Figure 3B). However, CBD significantly reduced AKT phosphorylation by approximately 40% in DU145 cells (p = 0.0006) (Figure 3C), suggesting that inhibition of AKT phosphorylation may contribute to the CBD-induced reduction in the expression of cell cycle proteins in these cells. These results support a recent report that CBD can inhibit AKT phosphorylation in the TRAMP cell line model of prostate cancer.¹² Our results suggest that in DU145 cells CBD may reduce cell viability by inhibiting the phosphorylation and activation of AKT, leading to downregulation of the cell cycle proteins CDK1, CDK2, and CDK4, induction of cell cycle arrest, and inhibition of cell proliferation. Alternatively, reduced AKT phosphorylation may occur downstream of the observed changes in cyclin and CDK expression.

A key finding from this study was the effect exerted by CBD on noncancerous prostate epithelial cells. To determine whether the effects of CBD on viability were cancer cell-specific, MTT analysis was conducted using two noncancerous androgen-sensitive prostate epithelial cell lines, PWR-1E and RWPE-1. Interestingly, the noncancerous cell lines were slightly more sensitive to CBD treatment than the cancer cell lines, with IC₅₀ values of 0.9 μM (PWR-1E) and 1.1 μM (RWPE-1) (Figure 4A), suggesting that the effects of CBD on viability are not specific to cancer cells. Fluorescence microscopy was used to determine whether the reduced cell viability occurred through an increased level of cell death or inhibition of cell proliferation. PWR-1E cells were treated with CBD (1, 2, or 10 μM) in the absence of serum. CBD significantly reduced the cell confluency at all doses tested. CBD also reduced nucleus area (p < 0.001) and circularity (p < 0.001), indicative of increased cell death (Figure 4B,C). Furthermore, CBD increased YO-PRO and propidium iodide positivity and reduced the fraction of healthy cells (Figure 4B,D). These results indicate that CBD induces cell death through increased apoptosis in noncancerous PWR-1E cells. The above findings contrast with previous studies reporting that CBD reduced cell viability in cell line models of colon cancer, breast cancer, and head and neck squamous cell carcinoma, without affecting the viability of corresponding noncancerous cell lines.^4,7,29,50 Furthermore, Sharma et al. reported that CBD inhibited the viability of prostate cancer cells with no significant effect observed in the prostate epithelial cell lines BPH-1 and PNT1B.¹⁰ Notably, both cell lines used in that study were grown with serum, which is likely to reduce the efficacy of CBD. Furthermore, all the above experiments were conducted following a shorter 24 h CBD treatment, and it is possible that a reduction in viability would be seen with longer treatments. In agreement with the current study, Deng et al. reported that 72 h CBD treatment reduced viability at similar potencies in glioblastoma cells and in noncancerous neural progenitor cells.⁵¹ Similarly, a recent study in cholangiocarcinoma showed that CBD effects were not specific to cancer cells.⁵² It should be noted that the current study used immortalized cell lines, which are artificially transformed to proliferate indefinitely, a process known to alter cellular properties including differentiation, DNA damage response, and chromosome structure.^53,54 Therefore, the observed cytotoxic effects may not necessarily translate to similar effects in true normal and healthy prostate cells. In fact, the doses used in the current study are well within the range that has been reported safe and well-tolerated in vivo. Several studies report that CBD doses up to 1500 mg/day are safe and well-tolerated in humans.⁵⁵ Moreover, cannabis-based medicines are currently approved for the treatment of various medical conditions and display low levels of toxicity. However, our findings suggest that the effects of CBD are not cancer cell-specific. A deeper investigation into receptor targets of CBD and effects of CBD on intracellular signaling pathways may provide greater insight into potential off-target effects and toxicity.

Activation of cell invasion and metastasis is another crucial step in cancer progression, and metastasis is the cause of approximately 90% of cancer deaths.⁵⁶ Thus, agents that can inhibit metastasis could play an important role in cancer treatment. Previous studies have shown that cannabinoids reduce cell invasion in several cancer types, including breast cancer, lung cancer, glioblastoma, and head and neck squamous cell carcinoma.^{3,6,50,57−59} In breast cancer, inhibition of cell invasion by CBD was accompanied by reduced secretion of the matrix metalloproteases MMP-2 and MMP-9.³ An additional study reported that CBD was capable of reverting the invasive mesenchymal phenotype of breast cancer cells to a noninvasive epithelial phenotype, accompanied by increased expression of the adherens junction protein E-cadherin.⁶ Here, we assessed the antimetastatic potential of CBD in prostate cancer. Treatment with a noncytotoxic dose of CBD significantly reduced the invasiveness of highly metastatic PC-3 cells by approximately 30% (p = 0.0003) (Figure 5A). CBD had no significant effect on DU145 cell invasion. To investigate the mechanisms underlying the observed CBD-induced reduction in PC-3 cell invasion, we measured the secretion of matrix metalloproteases and the expression of E-cadherin. CBD treatment did not alter the secretion of MMP-1, MMP-3, or MMP-9 (Supplementary Figure S5). Interestingly, treatment with a noncytotoxic dose of CBD induced a greater than 2-fold increase in E-cadherin expression in PC-3 cells (p = 0.0374) (Figure 5B). These results indicate that the CBD-induced reduction in PC-3 invasiveness may occur through increased expression of the cell adhesion protein E-cadherin, suggesting that CBD may promote a noninvasive epithelial phenotype in prostate cancer cells. To our knowledge, these findings provide the first evidence that plant-derived cannabinoids have anti-invasive activity in prostate tumor cells.

It is important to note that the above phenotypic screening was conducted using artificial 2D cell line models. Further assessment of the effects of CBD in more physiologically relevant models, for example, 3D cell culture models or animal models, will determine the likelihood of the observed phenotypic effects translating to therapeutic benefits in a clinical setting.

In conclusion, this study provides novel insights into the phenotypic effects and underlying mechanisms of action of the plant-derived cannabinoid CBD in prostate cancer. CBD inhibits the viability, proliferation, survival, and invasiveness of prostate cancer cells. CBD-induced inhibition of proliferation was associated with reduced expression of the cell cycle regulators cyclin D3, CDK4, CDK2, and CDK1 and reduced phosphorylation of the protein kinase AKT. The anti-invasive effects of CBD were accompanied by increased expression of E-cadherin. Further research is needed to identify the receptor target(s) of CBD in prostate cancer cells, to gain a deeper understanding of the mechanisms of action, and to investigate the effects of CBD on noncancerous cells. Additionally, testing of CBD in more biologically relevant models will determine whether the promising effects observed in vitro are likely to translate to therapeutic benefits. Overall, our findings indicate that CBD may have potential as a future chemotherapeutic agent in prostate cancer.