Colorectal cancer (CRC) is one of the leading causes of cancer-related death, with a rate of incidence that is remarkably increasing worldwide [1
]. It was estimated that 147,950 individuals would be newly diagnosed with CRC in the US in 2020, with 17,930 new cases (12%) in individuals aged younger than 50 years [2
]. Similarly, CRC has been placed at the second position in the oncologic death ranking for European countries in 2018 [3
]. Risk factors include lifestyle, diet, genetic factors, alterations in gut microbiota, a history of inflammatory bowel disease (IBD) and other additional pathologies (e.g., diabetes, obesity).
Palmitoylethanolamide (PEA) is an endogenous fatty acid amide belonging to the family of acylethanolamides (NAEs), which also includes anandamide (AEA) and oleoylethanolamide (OEA). PEA exerts anti-inflammatory, analgesic and neuroprotective properties with a multitarget mechanism, mostly mediated by peroxisome proliferator-activated receptor α (PPAR-α) [4
PEA improves inflammation in murine [5
] and human colon tissues [7
], which could be suggestive of a possible role for PEA in intestinal cancer, in light of the well-established association between intestinal inflammation and carcinogenesis [8
]. Furthermore, PEA has been shown to: (i) slow down melanoma cell survival in vivo and in vitro [9
]; (ii) induce cell death in high-grade astrocytoma/neuroblastoma cells [10
]; (iii) be decreased in human brain tumor tissues compared with healthy brain areas [11
] and (iv) potentiate the cytotoxic effect of anandamide on human breast cancer cells [12
]. Finally, PEA’s targets (e.g., PPAR-α, TRPV1 and CB receptors) are involved in a number of carcinogenesis mechanisms [13
]. However, the knowledge on PEA’s effects in colon carcinogenesis is still largely fragmentary, with only one study reporting the antiangiogenic effects of PEA on a human colon carcinoma cell line mediated by VEGF downregulation [14
Considering the paucity of data on PEA and CRC, this study is aimed at covering this gap by assessing PEA’s effects on colorectal cancer cell proliferation, migration and cell cycle as well as evaluating its possible in vivo effects in a murine model of colon cancer.
Taking into account the epidemiological data reporting a high rate of deaths related to colorectal cancer (CRC), there is a clinical need for new pharmacotherapeutic options. While PEA’s role in other cancers (e.g., melanoma, breast cancer, neuroblastoma) has been previously documented [9
], its role in CRC is still at a very early stage of understanding [14
]. In this study, by investigating um-PEA’s effects on cell proliferation, cell cycle and migration of CRC cell lines as well as in a murine model of colon cancer, we have provided key proofs of the beneficial effects of PEA in colon carcinogenesis. In preliminary experiments, um-PEA’s effect on cell proliferation was evaluated during a time period of 96 h incubation. Because the antiproliferative effect, at least for the 30 µM concentration, was maximal after 24 h of treatment, we selected this timepoint for further evaluations. The gradual disappearance of um-PEA’s effect after 24 h incubation could be due to its enzymatic catabolism [22
], since NAAA (i.e., the main PEA hydrolytic enzyme) is expressed in HCT116 cells. Also, the possibility that HCT116 cells could activate biological pathways able to overcome um-PEA’s antiproliferative activity cannot be ruled out.
Here, we have shown that um-PEA exerted antiproliferative effects in two different colon adenocarcinoma cell lines. The antiproliferative effect of PEA on Caco-2 cells has been previously documented [14
], with a maximal effect higher than that found in our experiments. Such a discrepancy, which might be due to several reasons, requires further investigations. Nevertheless, our results are in agreement with those reporting the antiproliferative effects of PEA on different tumor cell lines such as melanoma, neuroblastoma and breast cancer cells [9
]. Also of relevance, discrepancies in the potency and efficacy of PEA have been previously documented [24
]. Of relevance, um-PEA altered neither the growth of healthy cells nor their cell cycle phases, indicating that the effect of um-PEA is tumor-specific.
In order to depict the mechanism of action behind um-PEA’s antiproliferative effects on tumor cells, we conducted pharmacological studies, in which selective antagonists of PEA’s main targets [15
] were combined with um-PEA. Indeed, it has been demonstrated that PEA directly or not activates PPAR-α, GPR55 [4
], transient receptor potential cation channel subfamily V member 1 (TRPV1) and cannabinoid (CB) receptors [4
]. Here, our data highlight that the antiproliferative effect of PEA in the CRC cells was counteracted by PPAR-α and GPR55 antagonists, suggesting that um-PEA’s antiproliferative behavior is mediated by such targets.
To further explore the effect of PEA on tumor cells, we investigated whether this amide was able to regulate cell cycle progression. We showed the first proof that um-PEA led to colon cancer cell cycle arrest in the G2/M phase, with a parallel decrease in the percentage of cells in the S phase. The G2/M transition is regulated by the cyclin B1/CDK1 complex, a required checkpoint for cell cycle arrest in this phase [16
]. We also found that um-PEA induced an increase in the expression of the cyclin B1/CDK1 complex (a required checkpoint for cell cycle arrest in the G2/M transition), which participates, at least in part, in cell cycle arrest in the G2/M phase. Among the other NAEs, only anandamide has been reported to affect tumor cell cycle [25
], to date. In order to further depict um-PEA’s mode of action, we also investigated the involvement of the mTOR signaling pathway, which is known to regulate tumor cell growth [17
]. In light of our data, in which we did not find any change in the expression of the key actors of the mTOR pathway upon um-PEA treatment, we exclude that this pathway is involved in um-PEA’s mode of action. Although our data are not in line with those reported by Sarnelli et al. on Caco-2 cells [14
], they are in agreement with literature data showing that the downstream pathways associated with the activation of PPAR-α and GPR55 (which are the main PEA targets involved in our study) in colorectal cancer are mTOR pathway-independent. Since the G2/M checkpoint is known to be arrested in response to DNA damage [26
], we also verified this feature. Our results highlight that um-PEA damages DNA, thus supporting the functional relationship between the cell cycle G2/M arrest and PEA-induced DNA damage.
It will be a specific aim of further investigations to study whether or not PEA affects cancer stemness, a well-known hallmark of cancer progression and growth.
In favor of gaining knowledge on PEA’s effects on colorectal cancer cells, we also investigated the ability of PEA to influence CRC cell migration. We found that um-PEA treatment decreased the migration rate of two CRC cell lines by diminishing the expression of MMP2, a member of the matrix metalloproteinases family, which is known to be implicated in different hallmarks of carcinogenesis (e.g., tumor growth and migration) [19
]. Obviously, further insights into MMP2 involvement would arise from experiments in which MMP2 is pharmacologically inhibited. Also of importance, um-PEA reduced the expression of tissue inhibitor matrix metalloproteinase 1 (TIMP1), a pivotal player in regulating the balance of matrix remodeling and in cell proliferation [20
], whose suppression is implicated in the decreased progression of colon cancer [24
]. Overall, our data support the hypothesis that um-PEA is implicated in cell migration and proliferation, possibly via downregulation of MMP2 and TIMP1.
Finally, we demonstrated that um-PEA exerted beneficial effects in a murine model of colon cancer induced by the administration of AOM. This model has been extensively used to study the mechanisms underlying human sporadic colon cancer as well as to evaluate drugs with potential chemopreventive effects [25
]. Importantly, although we performed a quantitative analysis only, without the support of immunohistochemistry, our data clearly demonstrate that um-PEA showed a chemopreventive effect, being able to significantly reduce ACF and tumor number formation and by showing a trend in reducing the number of polyps. ACF, as well as polyps, are well-known precursors of colon cancer in humans [27
]. The chemopreventive effects of um-PEA could be related to its ability to attenuate in vivo colonic inflammation [5
], a well-known risk factor for the development of colon cancer [8
4. Materials and Methods
Ultramicronized PEA (um-PEA, powder particle size <10 µm, with the following distribution: <6 µm, 99.9%; <2 µm, 59.6%; <1 µm, 14.7%; <0.6 µm, 2%, as described in patent EP2475352 A1, with text from patent WO2011027373A1) was kindly provided by Epitech Group (Saccolongo, Italy). Azoxymethane (AOM) was obtained from Sigma-Aldrich (Italy). All reagents for cell cultures were purchased from Sigma-Aldrich (Milan, Italy), Bio-Rad Laboratories (Milan, Italy) and Aurogene Srl (Rome, Italy). The vehicles used for in vivo (ethanol/Tween20/saline in a ratio of 1:1:8, 2 mL/kg) and in vitro (0.1% ethanol) experiments had no effects on the responses under study.
4.2. Cell Lines
The human colon adenocarcinoma cell lines (i.e., HCT116 and Caco-2) (ATCC from LGC Standards, Milan, Italy) and the immortalized healthy human colonic epithelial cells (HCEC), derived from human colon biopsies, kindly gifted by Fondazione Callerio Onlus (Trieste, Italy) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, Milan, Italy) supplemented with 10% heat-inactivated FBS (Sigma-Aldrich, Milan, Italy). Cell lines were maintained at 37 °C in a humidified incubator with 5% CO2, and their viability was evaluated by trypan blue exclusion.
4.3. BrdU Incorporation
HCT 116, Caco-2 and HCEC were seeded in 96-well plates (1.0 × 104
cells per well), allowed to adhere (within 24 h) and starved by serum deprivation for 18 h. Tumoral and no-tumoral cells were all treated with um-PEA (1–30 μM). After 24, 48 and/or 72 h of treatment, pulsing cells were incubated with BrdU (10 µM) in the cell medium for 2 h. Thereafter, the proliferation of the cells was determined by using the BrdU proliferation ELISA kit (Roche, Milan, Italy) according to the manufacturer’s instructions. Using this assay, the antiproliferative effect of um-PEA (used at the submaximal concentration 10 μM) was also evaluated (in HCT116 cells) in the presence of GW6471 (3 μM, PPARα antagonist); ML191 (1 μM, GPR55 antagonist); 5′-iodoresiniferatoxin (0.1 μM, TRPV1 antagonist); AM251 and AM630 (both 1 μM, CB1 and CB2 antagonist, respectively) [15
] [all from Tocris, Rodano, Italy and incubated 30 min before um-PEA (10 μM)]. All results are expressed as a percentage of cell proliferation (n
= 4 experiments including 4 replicates for each treatment).
4.4. Scratch Assay
Sub-confluent HCT116 and Caco-2 cell lines were trypsinized and plated on a 2-well culture-insert (ibidi GmbH, Gräfelfing, Germany) inserted on a 24-well plate (5 × 104 cells/70 µL) and left to adhere overnight. After this time, the insert was removed, and cells were washed with phosphate buffer saline (PBS 1×) and treated with mitomycin C 30 µg/mL (Sigma-Aldrich, Milan, Italy) in serum-free media, in order to inhibit cell proliferation completely. After 2 h, tumoral cells were treated with um-PEA (30 µM) for 24 h. Wound area recovery was observed under a phase-contrast microscope (Leica, Wetzlar, Germany) and photographed at the time zero point (right after the mitomycin C removal) and after 24 h of treatment. Successively, by using the ImageJ software, the size of the opened area was measured from the digital images. The results are expressed as % of scratch closure (time zero/time 24 h × 100). Two images were acquired for each well, and at least 3 replicates were analyzed for each treatment. Four independent experiments were independently carried out.
4.5. Cell Cycle Analysis
Cell cycle analysis was performed according to BD Pharmingen™ BrdU Flow Kit (BD Biosciences, San Jose, CA USA) and conducted on HCT116 cells (1.5 × 105 cells seeded in a 6-well plate), overnight serum deprived and treated or not with um-PEA (30 μM) for 24 h. Cells were revealed by using BriCyte flow cytometer (Mindray, Trezzano sul Naviglio Italy), gated based on forward and side scatter to separate debris, and then the cellular events were further gated based on their BrdU and 7-AAD content. Data were analyzed by FlowJo v10 software (Tree Star, Ashland, OR, USA) and expressed as fold change of the cells in each cell cycle phase.
4.6. Gene Expression Analysis by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR)
mRNA obtained from human cell lines was extracted by using Purezol Reagent (Bio-Rad, Milan, Italy), following the manufacturer’s instructions. Reverse transcription was performed by using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), and qPCR was completed using SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) and gene specific primers, as detailed in Table 1
. All the data were normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the relative abundance was expressed by using the 2-ΔCt formula.
4.7. Western Blot Analysis
Western blot analysis was performed to investigate the expression of cyclin B1, CDK1, MMP2 and TIMP1 in the HCT116 tumoral cell line alone or in the presence of um-PEA (30 μM, 24 h). Cells were lysates in RIPA buffer (10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 140 mM NaCl, 1% Triton X-100, 0.1% (v/v) sodium deoxycholate, 0.1% SDS) supplemented with protease (Roche, Monza, Italy) and phosphatase inhibitors (Sigma-Aldrich, Milan, Italy). Forty micrograms of protein extract was fractionated by 12% SDS-PAGE according to the manufacturer’s protocols (Bio-Rad, Milan, Italy). After incubation with 5% (w/v) non-fat milk in TBS-T (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% (v/v) Tween-20) for 60 min, the membranes were incubated overnight with anti-Cyclin B1 (1:2000, Cell Signaling, Danvers, MA, USA), anti-CDK1 (1:1000, Invitrogen, Carlsbad, CA, USA), anti-MMP2 (1:1000, Invitrogen, Carlsbad, CA, USA) and anti-TIMP1 (1:200, Invitrogen, Carlsbad, CA, USA), and thereafter, anti-mouse IgG secondary antibodies (1:3000, Cell Signaling, Danvers, MA, USA), linked to horseradish peroxidase, were added. The signal was visualized by enhanced chemiluminescence using Chemidoc XRS (Biorad, Milan, Italy) and analyzed using Image Lab version 6.10.7. α-tubulin (1:1000, Cell Signaling, Danvers, MA, USA) was used as housekeeping normalizing protein.
4.8. DNA Fragmentation Assay
HCT116 cells were seeded in 10 cm culture dishes (5 × 105 cells per well), allowed to adhere (within 24 h) and starved for 18 h. Then, cells were treated with vehicle or um-PEA (30 µM). After 24 h of treatment, cells were washed twice in PBS, and incubated in DNA-lysis Buffer (50 mM Tris pH 7.5, 100 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 1% sodium dodecyl sulphate, 0.5 mg/mL proteinase K) for 1 h at 55 °C before extraction with phenol/chloroform/isoamyl alcohol. The suspension was then centrifuged (4000 rpm) for 5 min. DNA was precipitated with 1 volume of 5 M NaCl and 2.5 volumes of 95% (v/v) ethanol. The isolated DNA was resolved on a 1.5% agarose gel containing GreenSafe DNA Gel Stain (Canvax, Cordoba, Spain) in 40 mM Tris-acetate-EDTA buffer with electrophoresis at 80 V for 30 min. DNA fragments were visualized and photographed under ultraviolet light using Chemidoc XRS (Biorad, Milan, Italy).
Six-week-old male CD1 background mice were purchased from Charler River (Sant’Angelo Lodigiano, Italy), fed ad libitum with standard food (Mucedola srl, Settimo Milanese, Italy) and housed in polycarbonate cages under a 12 h light/12 h dark cycle at the Department of Pharmacy, University of Naples Federico II. All mice were used after a 1-week acclimation period (temperature 23 ± 2 °C; humidity 60%, free access to water and food). All the experimental procedures and protocols were in conformity with the principles of laboratory animal care, in compliance with national (Direttiva 2010/63/UE) laws and policies and approved by the Italian Ministry of Health. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals [28
4.10. Azoxymethane (AOM) Murine Model of Colon Cancer
The effect of um-PEA was evaluated in a murine model of chemically AOM-induced colon cancer. AOM (40 mg/kg in total, intraperitoneally (ip) was administered in mice at the single dose of 10 mg/kg once per week for four weeks. Um-PEA at a dose of 10 mg/kg was given (ip) three times per week for all the duration of the experiment, starting one week before the first administration of AOM in order to appreciate its chemopreventive effect. Um-PEA dose was selected on the basis of previous published work which showed the in vivo selective pharmacological effect of PEA in the intestine [5
]. All mice were humanely euthanized 12 weeks after the first injection of AOM. Based on our laboratory experience, this time (at the used dose of AOM) was associated with the occurrence of a significant number of aberrant crypt foci (ACF, which are considered preneoplastic lesions), polyps and tumors [31
]. Detection and quantization of ACF, polyps and tumors on the colon were performed as previously reported [32
4.11. Statistical Analysis
Data are expressed as mean ± SEM of n experiments. To determine statistical significance, Student’s t-test was used for comparing a single treatment mean with a control mean, and a one-way ANOVA followed by a Tukey multiple comparisons test and/or Dunnett’s multiple comparisons test was used for the comparison of multiple groups. Two-way ANOVA was used to compare different concentration–effect curves. A p-value < 0.05 was considered to be significant. G Power was used for sample size calculation.