Nowadays, there is an increasing interest in nutritional interventions because of their potential role in preventing colon tumorigenesis. Colorectal cancer is the third most prevalent cancer type in males and the second in females worldwide, displaying an increasing incidence even in traditionally low-risk countries such as Spain. In the last years, intervention strategies boosting the activity of the immune system are preferable, together with vehiculation of bioactive components with proven anticancer efficacy. Promotion of proper intestinal mucosal barrier function, cellular defense, and local or systemic inflammation are the main targets of these interventions [1
]. Impairment of the regulatory processes of these aspects represent a potential risk of enhanced growth and metastatic ability of cancer cells due to immune dysfunction [2
]. In the clinical setting, formulas based on different nutrients (i.e., arginine, glutamine, nucleotides, and/or ω-3 fatty acids) have demonstrated benefits in surgical patients by mean of the reduction of inflammatory responses and/or infections [4
]. These beneficial effects have been recognized by ESPEN (European Society for Clinical Nutrition and Metabolism) [7
] and ASPEN (American Society of Parenteral and Enteral Nutrition) [8
]. A meta-analysis has also demonstrated their effectiveness in diminishing inflammation and enhancing host immunity, although benefits in the clinical outcome were inconsistent [9
In this scenario, nutritional bioactive co-adjuvants can also help by influencing innate immune signals, which stem at the intestinal level to drive an adequate maturation of adaptive immunity. Nutritional interventions, in combination with classic pharmacological cancer treatments, represent a promising strategy to increase the number of patients which could respond successfully against immune–metabolic-based diseases derived from chemotherapy. The clinical seek for agents with favorable tolerability and safety profiles which could be incorporated as co-adjuvant players to pharmacological anticancer treatments makes also attractive the bioactive supplements. In cancer patients, activation of type-1 innate and acquired immunities are crucial for tumor immunotherapy to overcome immunosuppression [10
]. Immune response(s) depends on a multitude of factors such as the offending agent, the microenvironment of the effector cells, and the host’s capacity to respond. Moreover, it must constantly be adaptive and be able to integrate responses from many different cell types. The first line of defense of the innate immune system is the gut mucosa, which not only acts as the primary barrier, but it can also drive adequate adaptive immune responses by influencing its maturation processes. In this sense, dendritic cells (DCs), monocytes (MC), macrophages (Mθ), and other antigen-presenting cells (APCs) are upstream of T helper (Th) responses. Thus, understanding how naturally-occurring preventive/therapeutic agents selectively influence tolerance and immune cell function can set the stage for a new generation of active cancer immunotherapies. The latter could represent a path forward to develop durable, long-lasting immune responses against cancer [11
Herein, we have investigated the immune-nutritional tolerability and safety of a supplement based on a Rosmarinus officinalis
supercritical fluid extract enriched in phenolic diterpenes (RE) and shark liver oil enriched in alkylglycerols (AKG) as a bioactive lipid vehicle [12
] to be used as a potential co-adjuvant in intervention strategies for specific patients suffering from colorectal cancer and/or immune disorders.
2. Study Design and Materials and Methods
2.1. Supplement Composition
As we have previously demonstrated, specific ranges of carnosic acid/carnosol composition of supercritical fluid Rosmarinus officinalis L
extracts (RE) display relevant antitumor effects in colon and breast cancers [13
]. Moreover, RE synergizes with 5-Fu and sensitizes 5-Fu-resistant colon cancer cells to this drug [14
]. However, due to the low bioavailability of the bioactive compounds present in plants [15
], and with the aim to obtain therapeutic benefits of these compounds in the clinical setting, we have developed a supplement of RE, approved for human use, together with a vehicle system based on bioactive alkylglycerols (AKGs), also approved for human use (PCT/ES2017/070263). The current strategy was developed to enhance bioavailability of RE and to potentiate the antitumor effects of RE with bioactive alkylglycerols. For this, 60 healthy volunteers were randomized to participate in the study: 30 received the study soft gelatin capsules (CR)—rosemary supercritical extract (11.25 mg of diterpene phenols) and shark liver oil (SLO) enriched in AKGs (150 mg)—and another 30 volunteers received the control capsules (CC). Detailed composition of the study (CR) and control capsules (CC) are indicated in Supplementary Table S1
. The study and control capsules had the same coating and appearance, and capsules were consumed once a day for six weeks. The randomization was performed blindly. The company that produced the product was the only one that knew which group corresponded to each product
2.2. Subjects and Study Design
The set-up of the clinical trial consisted of a six week, double-blind, randomized and parallel pilot study with two study arms—RE and alkylglycerol containing capsules (CR) and control capsules (CC)—to evaluate the immunomodulatory effect (by quantifying changes in peripheral blood leukocyte subpopulations and the cytokine profile produced by peripheral blood mononuclear cells (PBMCs) after the ex vivo lipopolysaccharide (LPS) stimulation) (primary outcome), together with the modification of oxidation markers and modulation of the expression of genes related to immune responses, inflammation, oxidative stress, lipid metabolism, and cancer in healthy volunteers. Moreover, association of selected genetic variants (SNPs) with the responses to the intervention were also analyzed (secondary outcomes).
The pilot study protocol was approved by the local Ethics Committee of the IMDEA Food Foundation (IMD PI017), and it was carried out in accordance with The Code of Ethics of The World Medical Association (Declaration of Helsinki). Written informed consent was obtained by all subjects prior to starting the trial. Clinical Trial Registry number: This trial was registered at clinical trials.gov as NCT03492086 http://clinicaltrials.gov/
. PATENT: (PCT/ES2017/070263).
Volunteers were recruited from the Campus of Cantoblanco and Parque Científico de Madrid, from the GENYAL Platform, and from the dissemination of the study through the IMDEA Food Foundation web page and other means of communication.
Of the 123 volunteers interested in participating in the study, 63 were excluded before randomization for not meeting the inclusion–exclusion criteria of the study, not attending the screening visits (V0), or because they decided not to participate due to distance or personal reasons. Inclusion criteria: Age between 18 and 55 years; adequate understanding of the study; and willingness to complete the entire treatment. Exclusion criteria: Body mass index (BMI) >30 kg/m2; diagnosis of diabetes mellitus (T2D), hypertension, dyslipidemia, or other cardiometabolic disorders; impaired cognitive function; diagnosed hepatic, renal, or cardiovascular disease; subjects with primary immunodeficiency disorders, consumption of drugs with influence on the immune system, or splenectomy; presence of other pathologies like asthma, food allergies, Crohn’s, myasthenia gravis, or lupus; consumption of vitamins, minerals, supplements of antioxidant extracts, or protein supplements in the two weeks prior to the start of the study; subjects treated with drugs affecting the lipid or glycemic profile (i.e., statins, fibrates, diuretics, corticoids, anti-inflammatories, hypoglycemic agents, or insulin) during the previous 30 days; consumption of anticoagulants or antiplatelet agents, cyclosporine, acetylsalicylic acid, antihistamines, or sedatives; hypersensitivity to rosemary, to its components or other members of the family of lipped plants, or to soybean as excipient of the capsules; allergy or hypersensitivity to fish; habitual smoking or high consumption of alcohol; pregnant or lactating women; and high-intensity physical exercise.
Due to the lack of previous studies carried out with the products under study, a pilot study was carried out with a sample size of 60 volunteers (control group–CC: 18 females and 12 males; and study group–CR: 18 females and 12 males). From this study, we intended to calculate the sample size of subsequent studies, taking into account the main efficacy variable—with a final 60 subjects. The randomization procedure was provided by the Biostatistics Unit of the IMDEA Food Foundation. It was carried out using a randomization table generated by Statistic Software R 2.15 version (www.r-project.org
, University of Auckland, Auckland, New Zealand.)
The randomization and masking data were strictly confidential. Once the study was completed, the data file was verified and, after performing the results analysis, the codes were opened to proceed with the interpretation of the same. All volunteers registered a complete nutritional survey: 3-day habitual food registry and questionnaires about physical activity, capsule tolerance, and capsule consumption.
Before the first study visit, a group CC volunteer decided to retire due to having problems to attend to, and another volunteer had to be withdrawn due to increased levels of liver enzymes in the baseline analytical group CC, so that the study was finally initiated by 58 volunteers (35 females and 23 males): 28 in group CC (17 females and 11 males) and 30 in group CR (18 females and 12 males). Throughout the study, one volunteer was withdrawn as a precaution since she had persistent headaches, which turned out to be in group CC, so 57 participants finished (27 in group CC and 30 in group CR). No exclusion was made prior to the analysis, since all volunteers registered a consumption ≥75% of the agreement.
Blood samples were drawn and anthropometric data, bioelectric impedance analysis (BIA), and vital constants were monitored at each visit: V1 (baseline parameters), V2 (control visit), and V3 (final parameters). Morning urine was also collected (V1, V3).
Primary outcome: The immunomodulatory effect of the intervention was assessed by analyzing: (i) The levels of cytokines (IL-1β, IL-2, -4, -5, -10, IFNγ, TNFα) produced by PBMCs after ex vivo LPS stimulation; (ii) the effect on the hematological profile and differential counts of leukocytes; and (iii) the phenotyping of leukocyte subpopulations. Secondary outcomes: (iv) Modification of parameters related to inflammation and oxidative stress; and (v) modulation of the expression of genes associated with immune and inflammatory responses, lipid profile, oxidative stress markers, and cancer (from a panel of 48 specifically-selected genes). Single nucleotide polymorphism (SNP) analysis was included to identify putative associations with the differential responses to the intervention. Figure 1
A shows a diagram of the study and Figure 1
B displays the study flowchart and main objectives.
2.3. Biochemical Analyses
Triacylglycerol (TG), total cholesterol (TC), low-density (LDL-C) and high-density (HDL-C) cholesterol concentrations were measured by routine laboratory (Laboratorio CQS, Madrid, Spain, which follows the UNE-ISO 15189:2007 directives) methods. Urea, creatinine, hepatic enzymes—glutamyl oxaloacetic transaminase (GOT), glutamic-pyruvatetransaminase (GPT), and gamma glutamyltransferase (GGT)—bilirrubin, and alkaline phosphatase (AP) were also determined. The concentration of LDLox was measured by sandwich enzyme-linked immunosorbent assay (ELISA) by using the monoclonal antibody mAb-4E6 (Mercodia AB, Uppsala, Sweden). Urinary thromboxane B2 (TBX2) and total isoprostanes were quantified by competitive ELISA (Enzo Biochem, Inc., New York, USA and Oxford Biomedical Research, Michigan, USA, respectively).
2.4. Isolation of Peripheral Blood Mononuclear Cells (PBMCs)
Subjects were instructed to fast overnight before each blood collection. Blood samples were collected in heparinized tubes (BD Vacutainer, Franklin Lakes, NJ, USA) at each visit between 08:00 and 10:00 to minimize circadian variations, which processed within 2 h of collection and used for PBMCs isolation. Isolation was carried out under sterile conditions to avoid monocytes activation. Briefly, whole blood was diluted (1:1) with phosphate buffer solution (PBS) and centrifuged by density gradient with Histopaque-1077 (Sigma–Aldrich, Madrid, Spain), according to the manufacturer’s instructions. After collection, PBMCs were washed twice with PBS.
2.5. Multiplex Bead Immunoassay
For the analysis of the ex vivo cytokine profile produced by PBMCs after LPS stimulation, isolated PBMC were first incubated for 12 h and then treated with LPS. Supernatants were recovered to determine concentrations of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IFNγ, and TNFα using a magnetic bead-based immunoassay (Human High Sensitivity T Cell Magnetic Bead Panel A MAGPIX-Luminex) kit from Millipore Iberica SA (Madrid, 28050, Spain), following the manufacturer’s instructions. A minimum of 50 beads per parameter were analyzed by the MAGPIX-Luminex system (Millipore Iberica SA (Madrid, 28050, Spain). Raw data (median fluorescence intensity, MFI) were analyzed with the xPONENT software 4.1 (Luminex system, Millipore Iberica SA (Madrid, 28050, Spain).
2.6. FACS (Fluorescence Activated Cell Sorting) Analysis of Cluster of Differentiation (CDs) Markers
Analysis of cluster differentiation markers was achieved by flow cytometry with a Gallios analytical flow cytometer (Beckman Coulter, Irving, TX, USA). Aliquots (100 µL) of peripheral blood were mixed with the following fluorochrome-conjugated antibodies: Anti-human CD3-APC, CD4-APCeF750, CD8-PC7, CD14-PC5.5, CD16-PB, CD45-KrO, CD54-FITC, and CD56-PE (Beckman Coulter, Irving, TX, USA), and lysed with the Beckman Coulter Versalyse solution following the manufacturer’s recommendations. To obtain the cell counts for the different populations 100 µL of Flow-Count Fluorospheres (Beckman Coulter, Irving, TX, USA) were added.
2.7. Cell Culture and In Vitro Treatments
Human colon cancer cells SW620 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). SW620 cells were cultured in DMEM supplemented with 10% of fetal bovine serum (FBS) and 2 mmol/L glutamine. Cells were maintained under standard conditions of temperature (37 °C), humidity (95%), and carbon dioxide (5%).
For the in vitro validation of the molecular targets found in the clinical trial, we treated SW-620 CRC cells with three different doses corresponding to 0.5 × IC50
, 1 × IC50
and 2 × IC50
for 4, being the IC50
value in this cell line of 36.46 ± 7.23 mg/mL as previously described [14
2.8. RNA Extraction
Total RNA was extracted and purified from homogenized PBMCs with miRNeasy minikit, (Qiagen, Valencia, CA, USA) following the manufacturer’s protocol. Recovered RNA’s concentration and integrity were verified using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technology, Cambridge, UK).
2.9. Gene Expression Analysis
After DNAse I treatment (Invitrogen, Madrid, Spain), 1 g of total RNA was reverse transcribed for 2 h at 37 °C with miScript®
II Reverse Transcription kit (Qiagen, Germantown, MD, USA), according to the manufacturer’s guidelines. A Taq-Man Low Density Array (Applied Biosystems, Madrid, Spain) was specifically designed for this experiment, including 47 selected genes (Supplementary Table S2
) linked to immune system, inflammation, oxidative stress, lipid metabolism, and cancer-associated genes. Gene-expression assays were performed in a HT–7900 Fast Real-Time PCR. GAPDH was used as endogenous control. RT-StatMiner software (Integromics®
Inc., Madison, WI, USA) was used to detect and determine the quality control and differential expression analyses. The Expression Suite Software (Life Technologies, Madrid, Spain) program was used to obtain the Ct data. The ΔCt (Ct gene-Ct GAPDH) was calculated, and then the relative expression (RQ) between visits was calculated (V3−V1) following the 2−ΔΔCt
2.10. DNA Extraction and Genotyping
Genomic DNA from each participant was isolated from 300 μL of total blood using the QIAamp DNA Blood Mini Kit (Qiagen Sciences, Inc, Germantown, MD, USA) and recovered in 100 μL of nuclease-free water. Concentration and quality were then measured in a nanodrop ND-2000 spectrophotometer (ThermoScientific, Waltham, MA, USA). Genotyping was performed using the QuantStudio 12 K Flex Real-Time PCR System (Life Technologies Inc., Carlsbad, CA, USA) with a TaqMan OpenArray plates. Sixty-four single nucleotide polymorphisms (SNPs) were selected on the basis of their known involvement in different parts of the pathogenic processes of inflammation, immune system, obesity, lipid metabolism, redox homeostasis, and cancer (Supplementary Table S3
) and its related phenotypes. The results were analyzed using the TaqMan Genotyper software (Life Technologies Inc., Carlsbad, CA, USA).
2.11. Statistical Analyses
Data were analyzed with the statistical program R Statistical Software version 2.15 (www.r-project.org
, University of Auckland, Auckland, New Zealand). Description of qualitative data was made by absolute frequencies and percentages and quantitative data by mean and standard deviation (SD), standard error of the mean (SEM), or 95% confidence intervals (CI), depending on distribution of the data. For comparison of quantitative data between the two groups, Student t
-test (parametric test) or Mann-Whitney U or Wilcoxon test (non-parametric test) was used. Chi-square test or Fisher’s test were used to compare proportions of qualitative data between the two groups. Two-way repeated-measures ANOVA was used to evaluate differences of the effect of time (visits), treatment (group), and interaction time x treatment. The p
-value was adjusted by sex, age, and BMI. When significant interaction was observed, post-hoc analysis and the Bonferroni correction were applied.
For the statistical analysis of genetic data, X2 test was used to establish the deviation of the genotypic frequencies in controls compared with those expected under the Hardy–Weinberg equilibrium. Three-way ANOVA was used to evaluate the interaction time x treatment x genotype.
All the statistical tests were considered bilateral and a p-value < 0.05 as significant.
This study demonstrates the safety and tolerability of a dietary supplement (CR) based on a supercritical rosemary extract (RE) and alkylglycerols used as bioactive vehicle. These safety features of the dietary supplement open new avenues to its potential use as co-adjutant in different diseases. Rosemary extracts (RE) have been shown to exhibit antitumoral effects, both in vitro and in vivo, in a wide range of cancer types [13
]. Individual components of RE, such as carnosic acid, carnosol, urosolic acid, and rosmarinic acid, have been described as bioactive agents, although the complete extract displays higher bioactivities due to the synergistic effects of the different compounds. Rosemary extracts synergize with several antitumor agents [14
] used in clinics, frequently not only enhancing their effects, but also inhibiting their metabolism and reducing the appearance of resistance [14
]. Notably, RE is already approved as an agent for human health by the FDA and the EFSA.
Solubility in the physiological media has been identified as a major drawback for bioactive compounds, which failed to display beneficial effects in clinical studies. Herein, shark liver oil (SLO) enriched in bioactive AKGs (i.e., stimulation of hematopoiesis and immunological defenses and antitumor and anti-metastatic activities [19
]) was selected as a bioactive lipid carrier of rosemary extract. AKGs were shown effective for improving bioavailability and effectivity of the extract [23
]. The anticancer effects of AKGs have been associated with the recruitment and activation of macrophages as a primary antitumor defense (innate immune system response) and to the increased production of cytokines such as IL-12 and IFN-γ, enhancing the cytotoxic activity of NK cells and cytotoxic T lymphocytes, together with an increase proliferation of pre-activated T cells and NK cells [24
]. These effects could be reflected in the significant increase of the monocyte, as well as cytotoxic T cell population (Figure 2
). This cellular adaptation supports the ex vivo increased IFN γ production by established PBMC primary cultures from patients receiving the CR (Figure 3
These effects can synergize with the TLR4 inhibition by carnosic acid [25
] from rosemary extract when used as complementary therapeutics. Also, the ‘redox’ control of TLR3 activation by antioxidants from rosemary can modulate an adequate immune system response towards a monocyte immunosurveillance phenotype [26
]. The molecular signaling mediated by TLR3 involves STAT3 inducing a Th1 type immune response and a MθM1 polarization of the CD56−
population. Although there are controversial results regardless the role of AKG or shark oil containing AKG in the promotion [27
] or not [28
] of a MθM1 phenotype, our results seem to indicate that the combination of RE with AKG could contribute to a more balanced M1/M2 of the Mθ population [29
]. These effects could involve the induction of IFN-γ production from naive and activated T and NK cells, enhancing the cytotoxic activity of NK cells and cytotoxic T lymphocytes (Figure 3
) favoring host defense and protection. Similarly, the association of IFN-γ with the CR group of treatment could predispose immune response(s) towards a more tough control of cell growth [30
]. The observed reduction in the expression of JAK1
supports the potential of CR to attenuate local inflammation in low grade inflammatory diseases (i.e., metabolic syndrome, obesity, and type 2 diabetes) that could predispose to cancer [32
In addition, NFE2L2
expression levels significantly diminished in the study group (CR) (p
= 0.031) compared to control group. Nrf2 is a transcription factor activated by oxidative stress, leading to the expression of antioxidant and detoxifying enzymes and drug transporters (MRPs), as well as anti-apoptotic proteins and proteasomes. Nevertheless, several reports support a role of Nrf2 in oncogenesis. Oncogenes K-Ras, B-Raf,
mediate the transcription and amplification of Nrf2 in cancer cells, contributing to the oncogenic process by diminishing ROS levels [34
]. Thus, the observed downregulation in the study group (CR) suggests an overall improvement towards a reduction of oxidative stress and/or inflammation. Moreover, in patients with cancer, downregulation of Nrf2 is expected to contribute to the reduction of drug resistance and an overall therapy improvement.
expression levels significantly decreased in the study group (CR) (p
= 0.008) compared to control (CC). BMP2 has recently been described to contribute to the promotion and development of colon cancer stem cells, together with an increase in drug resistance. Thus, the nutritional intervention based of RE-AKG (CR) suggests a positive influence on controlling the pool of colon cancer stem cells (CSC) [35
expression levels also decreased in the study group (CR), although not significantly (p
= 0.083). Chokα is a well described oncogene which stimulates proliferation, transformation, invasion, and metastasis, and it also potentiates tumorigenesis of other oncogenes such as RhoA [36
]. It is overexpressed in different human tumors, such as breast, lung, prostate, and colon cancer, and it has been proposed a biomarker with prognostic value of the evolution in early stages of non-small-cell lung (NSCL) cancer [37
]. Moreover, Chokα activity has been shown to be increased in colon adenocarcinomas relative to normal tissue, and it has been proposed as an important target in cancer therapy [38
]. Thus, the observed decrease in the study group (CR) compared to control support its benefits in patients with cancer.
Importantly, the observed results in gene expression regulation were also confirmed in SW-620 colon cancer cells treated only with RE, reflecting the specific role of the phytochemicals present in the RE extract.
The association between different genetic variants (single nucleotide polymorphisms, SNPs) and the response to the dietary intervention was analyzed in 64 single nucleotide polymorphisms (SNPs), which have been related to inflammation, immune system, obesity, lipid metabolism, redox homeostasis, and cancer. Given the small sample size to perform the evaluation by genotypes, the results were analyzed by the dominant model.
Study group homozygous carriers for the major allele (CC) of IFNγ rs2069727 SNP, displayed significant reduced levels of IL-5 (p = 0.000361). Study group (CR) volunteers genotyped AG+AA (carriers of the minor allele) for ALOX5 rs7913948 SNP displayed significant reduced levels of IL-5 (p = 0.004827). A significant interaction was also found between FABP2 rs1799883 SNP and absolute CD45+ counts. Study group carriers of the minor allele (CT, TT) for FABP2 rs1799883 SNP displayed significant increased numbers in absolute cell counts for CD45+ marker. To the best of our knowledge, there is a lack of studies in relation to the biological significance of these findings. These results reflect the genetic contribution to the differential responses to this nutritional intervention and warrants further attention and future studies.
One of the main limitations of the study is the age and healthy status of the volunteers, as impaired immune function has been implicated in the declining health and higher incidence of cancer in the elderly [40
]. Nevertheless, as shown in this study, the proposed underlying mechanism(s) seems to imply innate immune ‘Toll-like’ receptors (TLRs) driving the polarization of myeloid cells. It has been suggested that TLRs signaling contributes to the pathogenesis of age-related neurodegenerative diseases [41
]. Thus, age-related changes compromise the first cellular line of defence, such as neutrophils and macrophages, as well as cytotoxic T lymphocytes activity. In this scenario, the regulatory influence of the developed product increasing a surveillance monocyte population it is not expected to cause ‘deleterious’ health effects. The impaired or improved efficacy of the product will be genetically and physiologically determined by age and physiological status of cancer patients, affecting bone marrow myeloid cell production and immune-senescence. This effect can result similar as that on chemotherapeutic agents, which efficacy or toxicity will be determined by metabolic capacities affected by age. Another limitation of this pilot study is the small sample size, and more studies are needed to confirm the results obtained in this study. These aspects warrant further studies to establish the efficacy of the product in the clinical practice.
In summary, our results support the safety and tolerability of a dietary supplement based on rosemary extract and alkylglycerols as vehicle that is reflected moderate changes in plasmatic leukocyte populations associated to the study intervention.
The lack of immunosuppressive effects, together with a polarized leukocyte population towards phagocytic and effector cytotoxic T cells in response to CR consumption, supports its inclusion in anticancer strategies. A summary of the potential main biological benefits of the RE-AKG-based formula found in this study that encourages its usefulness as co-adjuvant in colon cancer therapy is presented (Figure 6