Cannabinoids are bioactive lipids able to interact with specific cell-surface cannabinoid receptors (CBs). They can be divided into three main classes: endocannabinoids (endogenous ligands), phytocannabinoids (derived from Cannabis Sativa
), and synthetic cannabinoids. In the last few decades, growing evidence has reported their ability to impair cancer progression both in vitro and in xenograft models of human cancers. The anti-tumorigenic activities include: (i) inhibition of cancer cells’ proliferation and migration, (ii) induction of cancer cell death, (iii) impairment of neoangiogenesis, and (iv) modulation of anti-tumor immune response [1
]. Δ-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are the two best-characterized active components contained in marijuana. THC binds with high affinity both CB1
, while CBD, which lacks psychotropic activity, mainly interacts with vanilloid receptor 1 (TRPV1). Of interest, CB1
were found up-regulated in malignant tissues compared to their non-transformed counterparts and a correlation between high expression and poor prognosis has been proven for different human tumors [4
Cannabinoids have been shown to be promising anticancer agents in prostate cancer treatment [5
]. In prostate cancer patients, high expression of CB1
has been associated with a worse prognosis [6
], and several authors reported CB1
-mediated anti-proliferative and anti-invasive effects of cannabinoids in prostate cancer cells [8
]. Furthermore, it has been recently demonstrated that CB2
also controls tumor cells proliferation, migration, and invasion in both prostate cancer cell lines and in vivo models [13
To date, it is widely accepted that cancer outcome does not only depend on the behavior of cancer cells, but also on the tumor microenvironment (TME) that coevolves with cancer cells, sustaining the enhancement of tumor malignancy. We previously demonstrated that cancer-associated fibroblasts (CAFs), the most represented stromal cells in the prostate TME, play an intriguing role during all stages of disease progression, including metastasis [14
]. Herein, we show a selective action of WIN 55-212.2 mesylate, a synthetic cannabinoid with affinity for CB1
higher than THC, on prostate cancer cell lines without affecting healthy counterpart. Furthermore, we demonstrated for the first time that patient-derived prostate CAFs upregulate CBs expression compared to normal fibroblasts (HPFs). WIN 55-212.2 mesylate strongly impaired CAFs’ activation and CAFs-induced tumor invasion. By using selective antagonists of CB1
, we proved that WIN 55-212.2 mesylate operated mainly through CB2
. Finally, we demonstrated that migration of androgen-independent prostate cancer cells (PC-3 and DU-145) and CAFs phenotype could be regulated by an autocrine self-sustaining loop of endocannabinoids.
Overall, these data support the use of cannabinoids as promising anti-cancer drugs in prostate cancer patients, since they are able to simultaneously strike cancer and stromal cells.
To date, an increasing number of preclinical studies have reported exciting anticancer properties of cannabinoid-related drugs, both in vitro and in xenograft models of several human cancers, including prostate cancer [1
]. Interestingly, cannabinoids have been shown to selectively target tumor cells, which upregulate CBs compared with their healthy counterparts. Furthermore, in non-transformed cells that constitutively express CB1
, cannabinoids modulate cell-survival and cell-death pathways differently than in tumor cells. The best-established example is that of glioma cells and astrocytes. In glioma cells, CB1
activation induces de novo ceramide synthesis, which correlates with inhibition of Akt survival pathway and cell death [34
]. On the contrary, in astrocytes CB1
-mediated pathway is associated with Akt phosphorylation and cell survival [37
]. This differential response has been reported also in rat thyroid epithelial cell lines and skin carcinoma cells [39
]. Our data now reinforce this outcome selectivity of cannabinoids, showing that WIN 55-212.2 mesylate at concentrations higher than 5 µM induces cell death in prostate cancer cell lines, both androgen-sensitive and insensitive, without affecting healthy prostate epithelial cells (Figure 1
a). Similarly, CBD 5 µM triggers cell death in cancer cell lines, while PNT-1 viability is not affected. It is not surprising that in response to cannabinoids’ concentration being higher than 30 µM, cell survival tends to increase in both healthy and cancer cells. Indeed, several studies have demonstrated that doses and the duration of the treatment are determinant factors to induce anti-neoplastic or pro-tumoral activity, which also depends on the agonist administered and the type of tumor [41
]. This effect might rely, at least in part, on cannabinoids’ capacity to form homo- or hetero-oligomers in response to various agonists, interfering with the activation of other G signaling proteins different from inhibitory G proteins normally coupled to CB1
]. However, the molecular basis of this “yin and yang” behavior remain an open question in cannabinoid field.
Since TME is now recognized as a hallmark of cancer, and stromal cells coevolve with tumor cells and sustain tumor progression [46
], assessing the effect of cannabinoids on the microenvironmental components is of exciting interest. Here, we showed for the first time that patient-derived CAFs upregulate CBs compared to HPFs isolated from the healthy region (Figure 2
a,b). This result is consistent with the increased expression of CB1
in HPFs in response to tumor-secreted inflammatory cytokines (Figure 2
c–e). In particular, HPFs and CAFs exhibit a different expression profile of CBs: CB2
expression is more than 2-fold higher in CAFs than in HPFs, CB1
is upregulated in CAFs, although to lesser extent, and TRPV1 is weakly expressed in both cell types. However, the treatment with WIN 55-212.2 mesylate does not affect cell survival, while CBD strongly impairs fibroblast viability (Figure 3
a). These results suggest that CBD-mediated cell death could be dependent on signalling from non-canonical CBs, such as PPAR-γ. In keeping with that theme, Ramer et al. recently demonstrated that CBD induces apoptosis in primary cultures and cell lines of lung carcinoma via cyclooxygenase 2 and PPAR-γ [47
]. Fibroblasts’ response to WIN 55-212.2 mesylate is supported by several studies reporting that the effect of THC and its analogues on cell survival is mediated by CB1
, while CB2
regulates its anti-inflammatory properties [9
]. We proved that treatment with WIN 55-212.2 mesylate strongly affects the CAFs’ phenotype and their ability to increase tumor cell invasiveness, via the CB2
receptor (Figure 3
b–g and Figure 4
). The controversial effect of CBD on activated stromal cells invasion should be further investigated to clarify its role on the modulation of stromal reactivity and the expression of other CBD sensitive receptors, and the role of CBD as an inverse agonist/antagonist should be evaluated. Our results are supported by previous studies demonstrating that cannabinoids are able to reduce inflammation and modulate wound healing processes in mouse models of autoimmune disorders [52
]. If we consider cancer as a state of chronic inflammation, our data strongly reinforce the use of cannabinoids as a next generation treatment for cancer.
Finally, it has been widely demonstrated that alterations in endocannabinoid system correlate with tumor onset and progression [1
]. Then, we wondered whether in addition to the inhibitory effect triggered by targeting CB1
with synthetic agonists, the blocking of endogenous system could affect cancer and stromal reactivity. Our results suggest that both migration abilities of androgen-insensitive prostate cancer cells and prostate CAFs reactivity could be in part sustained by an autocrine self-sustaining loop of endocannabinoids (Figure 5
). Interestingly, the involvement of CB2
has been demonstrated in tumor and stromal cells, as observed by the decrease of both cancer cell migration and CAFs α-SMA expression following the treatment with the selective antagonist JTE-907. In contrast, the treatment with AM281, the selective antagonist of CB1
, showed, in tumor cells, opposite effects at 0.5 and 1 µM, suggesting that the biological effects of CB1
impairment could be dependent on the concentration and cell type, as already reported in the literature. Indeed, Endsley et al. showed that 2-AG may exert anti- and pro-invasive properties on prostate cancer cells depending on concentration [10
]. They showed that autocrine 2-AG exhibited an anti-invasive effect in androgen-independent prostate cancer cells [10
]. In contrast, when 2-AG was exogenously added at 1 µM, PC-3 and DU-145 cells invasiveness significantly increased [12
]. Accordingly, both endogenous CB1
agonists and antagonists may exert different effects depending on the concentration. Further studies will be needed to clarify this response.
Overall, our results stress the importance of the endocannabinoid system in prostate cancer progression and reinforce the therapeutic potential of WIN 55-212.2 mesylate in prostate cancer treatment, since it is able to simultaneously impact on cancer cells and stromal compartments. Further studies will be needed to clarify the role of endocannabinoids in tumor progression and aggressiveness in order to develop new possible therapeutic approaches to counteract tumor progression.
4. Materials and Methods
4.1. Antibodies and Reagents
For the western blot analysis, the following antibodies were used: anti- α -SMA (#number A5228) from Sigma-Aldrich (Darmstadt, Germany); anti-CB1 (#number ACR-001) and anti-TRPV1 (#number ACC-030) from Alomone Labs (Jerusalem BioPark (JBP), Jerusalem, Israel); anti-CB2 (#number sc-293188), anti-GAPDH (#number sc-365062), and secondary antibodies from Santa Cruz Biotechnology (Dallas, TX, USA); anti-ERK1/2 (#number 9102), pERK1/2 (#number 9101), SMAD2/3 (#number D7G7) and pSMAD2/3 (#number D27F4) from Cell signaling (Danvers, MA, USA). All primary antibodies, except for anti-CB1 (1:500), were used at 1:1000. CBD (#number 90899) was purchased from Sigma-Aldrich (Darmstadt, Germany), and WIN 55-212.2 mesylate (#1038) was from Tocris Bioscience (prior Ministerial Decree SP/096, released on September 11th 2018) (Bristol, UK). Recombinant TGF-β 1 (#number 130-095-067) was provided from Miltenyi Biotec (Bergisch Gladbach, Germany), human TNF-α (#number 300-01A) and IL-6 (#number GMP200-06) were from PeproTech (London, UK). TGF- β receptor inhibitor (A8301, #number 2939), and CB1 and CB2 antagonists (AM281, #number 1115 and JTE-907, #number 2479, respectively), were from Tocris Bioscience (Bristol, UK). Blocking antibodies for IL-6 (α-IL-6, #number mabg-hil6-3) were from InvivoGen (San Diego, CA, USA). Matrigel™, Basement Membrane Matrix (#number 356234) was from BD Biosciences (Allschwil, Swiss).
4.2. Cell Models and Cell Cultures
LNCaP and PNT-1 cells were purchased from Sigma-Aldrich (Darmstadt, Germany); PC-3 and DU-145 cells were from the American Type Culture Collection (ATCC, Manassas, VA, USA). PC-3 cells were grown in HAM’S F12 medium; DU-145 cells were cultured in DMEM, LNCaP and PNT-1 cells were cultured in RPMI 1640. Human prostate fibroblasts were isolated from aggressive prostate cancer-bearing patients (Gleason score 4+5, 4+4, grade ≤ pT3), with HPFs and CAFs deriving from the healthy region and the intra-tumoral area, respectively. The isolated HPFs and CAFs were maintained in DMEM. The patients enrolled in the study underwent prostatectomy without receiving prior hormone deprivation therapy. All the enrolled patients provided their signed informed consent, in agreement with the Ethics Committee of the Azienda Ospedaliera Universitaria Careggi (approval code: CEAVC – 2018-256, on October 10th 2018). All the cells used were grown at 37 °C/5% CO2. All cultured media were purchased from EuroClone (Milano, Italy) and supplemented with 10% fetal bovin serum (FBS, EuroClone, Milano, Italy), 1% penicillin/streptomycin and 2 mM glutamine (Sigma-Aldrich, Darmstadt, Germany).
4.3. Conditioned Media Preparation
Conditioned media were obtained from sub-confluent cells maintained in serum-free medium (St Med) for 48 h. Once collected, conditioned media were filtered and used freshly or frozen at −80 °C until use.
4.4. Western Blot Analysis
Cells were lysed in Ripa buffer (Thermo Fisher, Waltham, MA, USA), after the addition of protease and phosphatase inhibitor cocktails (#number P1860 and #number 026M4058V, Sigma-Aldrich, Darmstadt, Germany), and total proteins were quantified with BCA assay (#number SLCC1765, Sigma-Aldrich, Darmstadt, Germany). Between 10 and20 µg of total protein lysate was loaded on each precast gel (4–20% acrylamide, mini-PROTEAN TGX Stain-Free, Bio-Rad, Hercules, CA, USA), and that was transferred onto a membrane by the Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA). The immunoblot was performed as previously described [14
] and was analyzed by Amersham Imager 600 (GE, Healthcare Life Sciences, Marlborough, MA, USA).
4.5. Crystal Violet Staining
HPFs and CAFs (3 × 104 cells) or prostate cancer cells (1 × 104 cells) were seeded in 24-multiwell plates and maintained in St Med for 24 h, before receiving WIN 55-212.2 mesylate or CBD, at concentrations ranging from 0.5 to 100 µM. After 24 h, cells were washed twice with PBS, fixed with formaldehyde 4% (v/v), and then incubated at room temperature with crystal violet solution (crystal violet 0.5% (w/v) and methanol 20% (v/v)). After the incubation, the cells were washed with PBS and treated with SDS 2% (w/v) for 1 h at 37 °C. The absorbance at 595 nm was measured.
4.6. Gelatin Zymography
Conditioned media from CAFs and HPFs activated in vitro by TGF- β 10 ng/mL, treated or not for 24 h with cannabinoids (WIN 55-212.2 mesylate and CBD 2.5 µM) and CBs antagonists (AM281 and JTE-907 0.5 µM), were collected, 10-fold concentrated with Amicon centrifugal filter (UFC800324, Merck-Millipore, Burlington, MA, USA) and analyzed by gelatin zymography as previously described [54
4.7. Boyden Chamber Assay
Invasion and migration assays were performed in transwells (8 µm pore polyvinylpyrrolidone-free polycarbonate filters, #number CC3422, Corning, Corning, NY, USA) with or without pre-coating with 50 µg/cm2
of reconstituted Matrigel, as previously described [27
]. Briefly, CAFs and HPFs activated in vitro by TGF- β 10 ng/mL, were serum-starved for 24 h and then treated with WIN 55-212.2 mesylate and CBD 2.5 µM for an additional 24 h. Then, 1 × 105
fibroblasts were added to the upper chamber of Matrigel-coated transwells and allowed to invade at 37 °C for 24 h towards complete medium (20% FBS).
PC-3 cells were incubated at 37 °C for 48 h with conditioned media from CAFs treated or not with WIN 55-21.2 mesylate and CBD 2.5 µM. Then, 3 × 104 cells were added to the upper chamber of Matrigel-coated transwells and allowed to invade at 37 °C for 16 h towards complete medium (10% FBS).
To investigate the effect of an endocannabinoids autocrine self-sustaining loop on cell migration, PC-3 or DU-145 cells were treated at 37 °C for 24 h with AM281 and JTE-907 at 0.5 µM and 1 µM. The pharmacological agents were administrated alone or in combination and added again during the migration assay. 3 × 104 PC-3 and DU-145 cells were allowed to migrate in transwells at 37 °C for 16 h towards complete medium (10% FBS).
The migration/invasion was reported as fold change (FC) compared with the starved control (Untr).
4.8. Statistical Analysis
Statistics were performed using Prism 8 (GraphPad Software, San Diego, CA, USA). Data were represented as the means ± SEM of at least three independent experiments. Statistical analysis was performed by Student’s t test or one-way analysis of variance (ANOVA) followed by post-hoc test. The significance was set at p value < 0.05.