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Cancer Prev Res (Phila). Author manuscript; available in PMC 2021 December 01.
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Published in final edited form as:

Cancer Prev Res (Phila). 2021 June ; 14(6): 619–626. doi:10.1158/1940-6207.CAPR-20-0585.

LEUCOSELECT PHYTOSOME MODULATES SERUM

EICOSAPENTAENOIC ACID, DOCOSAHEXAENOIC ACID and
PROSTAGLANDIN E3 IN A PHASE 1 LUNG CANCER
CHEMOPREVENTION STUDY
Jenny T. Mao1, Bingye Xue1, Sili Fan2, Patricia Neis1, Clifford Qualls3, Larry Massie4, Oliver
Fiehn2
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1Pulmonary,Critical Care and Sleep Section, New Mexico Veterans Administration Health Care
System, and University of New Mexico.
2UC Davis Genome Center.
3Biostatistics,
Biomedical Research Institute of New Mexico, New Mexico Veterans Administration
Health Care System, and University of New Mexico.
4Pathologyand Clinical Laboratory Services, New Mexico Veterans Administration Health Care
System, and University of New Mexico.

Abstract
Grape seed procyanidin extract (GSE) has been shown to exert antineoplastic properties in
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preclinical studies. Recently we reported findings from a modified phase I, open-label, dose-
escalation clinical study conducted to evaluate the safety, tolerability, maximum tolerated dose
(MTD), and potential chemopreventive effects of leucoselect phytosome (LP), a standardized GSE
complexed with soy phospholipids to enhance bio-availability, in heavy active and former
smokers. Three months of LP treatment significantly decreased bronchial Ki-67 labeling index
(LI), a marker of cell proliferation on the bronchial epithelium. Because GSE is widely used as a
supplement to support cardiovascular health, we evaluate the impact of oral LP on the fasting
serum complex lipids metabolomics profiles in our participants. One month of LP treatment
significantly increases eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the
omega-3 polyunsaturated fatty acids (n-3 PUFA)s with well-established anti-cancer properties. LP
also significantly increases unsaturated phosphatidylcholines (PC), likely from soy phospolipids in
the phytosome and functioning as transporters for these PUFAs. Furthermore, 3 month of LP
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treatment significantly increases serum prostaglanding (PG) E3, a metabolite of EPA with anti-
inflammatory and antineoplastic properties. Such increases in PGE3 correlate with reductions of
bronchial Ki-67 LI (r = −0.9, p = 0.0374). Moreover, post-treatment plasma samples from trial
participants significantly inhibit proliferations of human lung cancer cell lines A549
(adenocarcinoma), H520 (squamous cell carcinoma), DMS114 (small cell carcinoma), and 1198

Address Correspondence to: Jenny T. Mao, M.D., Pulmonary, Critical Care and Sleep Section, New Mexico VA Health Care System,
1501 San Pedro Drive. SE, Albuquerque, New Mexico, Phone: (505) 265-1711, ext 4509, FAX: (505) 256-5751, [email protected].
Conflict of Interests: The authors declare that there is no conflict of interests.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed
 Mao et al. Page 2

(preneoplstic cell line). Our findings further support the potential utility of LP in reducing
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cardiovascular and neoplastic risks in heavy former and active smokers.

Keywords
Grape seed procyanidin extract; eicosapentaenoic acid (EPA); docosahexaenoic acid (DHA); PGE3
and unsaturated phosphatidylcholines

INTRODUCTION
Preclinical studies demonstrate various antineoplastic effects of GSE against lung cancer (1–
5). To faciliate clinical translation, we have selected an inexpensive, over the counter GSE
preparation (leucoselect), standardized to smaller size oligomeric procyanidins (OPC) and
complexed with soy phospholipids into phytosomes to improve bioavailability, for
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translation into a phase I lung cancer chemoprevention trial in heavy former or active
smokers at high risk for lung cancer. This leucoselect phytosome (LP) has been shown to
improve oxidative status, including the total antioxidant capacity of plasma, and reduce LDL
susceptibility to oxidative stress in heavy smokers (6). We have recently reported the
feasibility of LP as a potential lung cancer chemopreventive agent from the study, including
a significant reduction of bronchial Ki-67, a marker of cell proliferation on the bronchial
epithelium and a key surrogate endpoint biomarker for lung cancer chemoprevention trials
(7).

Because GSE is widely used to promote cardiovascular health, we evaluate the effects of
oral LP on the profiles of systemic complex lipid metabolomics by comparing matched pre-
and post-treatment fasting serum samples from participants. LP treatment significantly
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increases serum EPA, DHA, and unsaturated PC. Furthermore, LP significantly increases
fasting serum PGE3, a downstream eicosanoid derived from EPA, at the end of 3 months of
treatment. Our findings support the continue investigation of LP for lung cancer prevention
and treatment.

MATERIALS AND METHODS

Leucoselect phytosome clinical study design
A single arm, dose escalation, modified phase I lung cancer chemoprevention study of 3
months of oral LP, comprised of standardized OPC complexed with soy phospholipid or
lecithin (1:2.6 w/w; Indena, Milan, Italy, supplied via Thorne Research), was conducted in
high risk heavy active or ex-smokers 21 years of age or older with a smoking history of at
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least 30 pack-years (pky) as previously described (7). Written informed consent was
obtained in accordance with the New Mexico VA Health Care System (NMVAHCS)
Institutional Review Board, following the guidelines of Declaration of Helsinki, Belmont
Report, and U. S. Common Rules. Qualified participants were treated with 1 capsule (cap),
450 mg/cap once a day, escalating weekly to 4 caps once a day for the rest of the treatment
duration as tolerated. Fluorescence bronchoscopy with bronchial biopsies were performed at
baseline and at the end of 3 months treatment. Serial fasting blood samples were collected at

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baseline, end of month 1 and end of month 3 treatment for comparative biomarker analysis.
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Blood samples were processed within 1 hour of collection, spun at 3000 rpm, 15 minutes for
serum and 10 minutes for heparin plasma, aliquoted into cryovials and stored at −80°C until
analysis.

Lipidomics by charged surface hybrid column (CSH)- electrospray (ESI) quadrupole time
of flight mass spectrometer (QTOF) tandem mass spectrometry (MS/MS)
Extraction: Serum was extracted using a previously described protocol (8). One organic
phase aliquot was re-suspended in 100 μL of methanol:toluene (9:1, v/v) mixture containing
50 ng/mL CUDA (12-[(cyclohexylamino) carbonyl]amino]-dodecanoic acid) (Cayman
Chemical). Samples were vortexed and sonicated for 5 min, centrifuged at 16,000 rcf and
prepared for lipidomic analysis. Method blanks and pooled human plasma
(BioreclamationIVT) were included as quality control samples.
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Chromatographic and mass spectrometric conditions for lipidomic reverse phase liquid
chromatography (RPLC)-high field quadrupole orbitrap mass spectrometer (QEHF)
analysis
Using an Agilent 1290 Infinity Ultra-High-Performance Liquid Chromatography (UHPLC)
system, re-suspended samples were injected at 3 and 5 μL for positive and negative ESI
modes, respectively, onto a Waters Acquity UPLC CSH C18 (100 mm length × 2.1 mm id;
1.7 μm particle size) with CSH C18 pre-column (5 mm × 2.1 mm id; 1.7 μm particle size).
The column was maintained at 65°C. To improve lipid coverage, different mobile phase
modifiers were used for positive and negative ESI mode analysis (9). For positive ESI mode,
10 mM ammonium formate and 0.1% formic acid were used; for negative ESI mode, 10 mM
ammonium acetate (Sigma–Aldrich) was employed. Both positive and negative ESI modes
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used the same mobile phase composition of (A) 60:40 v/v acetonitrile:water (LC-MS grade)
and (B) 90:10 v/v isopropanol:acetonitrile. The gradient started at 0 min with 15% (B), 0–2
min 30% (B), 2–2.5 min 48% (B), 2.5–11 min 82% (B), 11–11.5 min 99% (B), 11.5–12 min
99% (B), 12–12.1 min 15% (B), and 12.1–15 min 15% (B). A flow rate of 0.6 mL/min was
used. For data acquisition, positively charged lipids such as PC, lysoPC, were analyzed
using an Agilent 6530 QTOF mass spectrometer at resolution R=10,000 while negatively
charged lipids such as free fatty acids and phosphatidylinositols were analyzed using an
Agilent 6550 QTOF mass spectrometer at resolution R=20,000.

Data processing using MS-DIAL

Untargeted lipidomic data processing was performed using MS-DIAL (10) for
deconvolution, peak picking, alignment, and identification. The public LipidBLAST MS/MS
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spectra database was used, validated by retention time and m/z matching to authentic
standards (11). Detected lipids were used for statistical analysis when they were positively
detected in at least 50% of all samples in each group. Data were normalized by the sum-
norm of all identified lipids (mTIC) (12), to scale each sample. Normalized peak heights
were then submitted to R for statistical analysis.

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Measurements of serum PGE3 and LTB5

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PGE3 and LTB5 levels in matched fasting serum collected pre- and post-treatment were
measured using specific enzyme-linked immunosorbent assay kit (MyBioSource, San Diego,
CA, USA per manufacturer’s instructions.

Cell Cultures
As models to evaluate the anti-neoplastic bioactivity of oral LP against lung cancer, the
human NSCLC cell lines A549, H520, small cell lung cancer cell line DMS114 (purchased
from ATCC; Manassas, VA., in 2019, 2015, and 2016, respectively), and the bronchial
preneoplastic cell line 1198 (generously provided by Dr. Andres Klein-Santos, Fox Chase
Cancer Center, Philadelphia, PA, received in 2013) were co-cultured in vitro with matched,
pre- and post-3 months treatment fasting heparinized plasma from 6 study participants.
Experiments involving A549 was initiated within 6 months of purchase and was not further
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authenticated. ATCC uses Short Tandem Repeat profiling for cell line authentication. H520,
DSM114 and 1198 cells were not further authenticated. Cells were last tested for
mycoplasma in 2017. Cells were maintained as monolayers in an atmosphere of 5% CO2 in
air at 370C in 25-cm2 tissue culture flasks containing cell line specific culture medium as
previously described (4, 13). Only Cells within passage 3–6 at 70–80% confluence were
used. Aliquots (100 μl) of 6 × 104 cells/ml of A549 cells, 10 × 104 cells/ml of H520 cells, 12
× 104 cells/ml of DMS114 cells, or 10 × 104 cells/ml of 1198 cells were plated in 96 well
plates and incubated at 370C for 2 h, followed by addition of 10, 10, 5, or 5 μl heparinized
plasma, respectively, and incubated for 44 hours. Cells were then subjected to PrestoBlue
cell viability/proliferation assay.

PrestoBlue Cell viability/proliferation Assay

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To quantify cellular proliferation in conditioned cells, PrestoBlue HS cell viability reagent

(Invitrogen, Eugene, Oregon) at 1/10th of cell culture volume was added to conditioned cells
per well then incubated for 20 minutes at 37°C, 5% CO2, to measure the reduction of the
reagent by metabolically active cells according to the manufacturer’s instructions.

Statistical Analysis
The effects of LP treatment on complex lipid metabolomics were determined by comparing
baseline values with those obtained at 1 month of treatment using Wilcoxon signed-rank test
to assess the difference between treatment effect on each compound. Benjamini-Hochberg
procedure (14) was used to control the false discovery rate. Fold changes, defined as median
average of post-treatment divided by the median average of pre-treatment, were calculated
for each compound. Chemical similarity enrichment analysis (15) was performed using the
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raw p-value and fold change of each compound. Kolmogorov-Smirnov test was used to test
the difference at compound set enrichment level. All statistical analyses were conducted
using R.

The effects of LP treatment on PGE3 and LTB5 and plasma co-cultures with human lung
cancer and preneoplastic cells were determined by comparing matched pre- and post-3
months treatment values from each of the 6 participants who completed 3 months treatment.
Fold or percent change of each biomarker from each participant was calculated first by

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normalizing matched post-treatment to baseline pre-treatment values, followed by paired t

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tests. Data were expressed as the mean ± SEM in all circumstances where mean values were
compared. Differences were considered significant when p <0.05. Pearson correlation
coefficients were computed for modulations of serum EPA, DHA and PGE3 with
modulations of bronchial Ki-67 by LP from these participants.

RESULTS
Effects of one month of oral LP treatment on complex lipid metabolomics profiles in
fasting serum
To determine the effects of oral LP treatment on the systemic metabolomics profiles of
complex lipids, matched pre- and post-1 month-treatment fasting serum samples were
compared. A total of 8 sets of paired samples were available for evaluation. Among the 23
clusters of compounds, 3 clusters were found to be significantly altered: 1) Unsaturated fatty
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acid (FA), 2) Unsaturated PC, and 3) Carnitine (Table 1A and Figure 1A).

Effects of one month of oral LP treatment on EPA, DHA, and unsaturated PC in fasting
serum
Key coumpounds significantly altered/increased by LP treatment in the fasting serum of
study participants include EPA and DHA in the unsaturated FA cluster. In addition, LP
treatment significantly increased a total of 3 unsaturated PC, with PC(36:5) A as the key
compound within that cluster. Moreover, plasmalogen PC(p-18:1/18:3) was also
significantly increased (Table 1B). Pearson correlation of modulations of EPA and DHA
with modulations of bronchial Ki-67 LI showed a trend toward statistical significance (Table
1C).
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Effects of 3 months of oral LP treatment on fasting serum PGE3 and LTB5

EPA can function as a substrate for cyclooxygenase (COX)s to synthesize unique 3-series
PG compounds, especially PGE3, which tends to have antiproliferative and anti-
inflammatory activities. To determine if the increase in systemic EPA by LP treatment might
lead to an increase in PGE3, the levels of PGE3 in matched pre- and post-treatment fasting
serum samples were measured. LP treatment significantly increased PGE3 levels by an
average of 45% (Fig. 1B). As EPA could be a precursor for LTB5, the levels of LTB5 were
also measured. LP treatment did not significantly change LTB5 levels. Increases of PGE3
significantly correlated with decreases of bronchial Ki-67 LI (Table 1C).

Post-treatment fasting plasma samples inhibit proliferations of human lung cancer and
preneoplastic cells
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To evaluate the systemic, antineoplastic effects from oral LP treatment, A549, H520,
DMS114 and 1198 human cell lines were co-cultured with matched pre- and post-3 months
treatment fasting plasma. Post-treatment plasma samples significantly inhibited cell
proliferations in comparison with pre-treatment plasma (Fig. 1C).

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DISCUSSION
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In this correlative study of our recently published, modified phase 1 lung cancer
chemoprevention study with LP in heavy former and active smokers, we demonstrate that
once a day oral LP results in significant increases of n-3 PUFAs EPA and DHA, unsaturated
PC, and PGE3 in fasting serum. Post-treatment fasting plasma samples also significantly
inhibit proliferations of human lung cancer and preneoplastic cell lines in vitro.

There are two major classes of PUFAs: the omega-3 (n-3) and the omega 6 (n-6). Three n-3s
have been most studied: alpha-linolenic acid (ALA, containing 18 carbon), EPA (20
carbons) and DHA (22 carbons). ALA is an essential FA that needs to be obtained from the
diet. ALA can be converted into EPA and then to DHA, but the conversion is limited
(reported rate of 15% primarily in the liver). Therefore, consumption of EPA and DHA from
foods or dietary supplements is required to practically increase their levels in the body. ALA
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is present in plant oils such as soybean, flaxseed and canola oils. Whereas DHA and EPA are
present in fish, fish oils and Krill oils, but are originally synthesized by microalgae, then
consumed by fish (16).

EPA and DHA are long chain n-3 PUFAs with anti-inflammatory and immunomodulatory
properties. They are believed to benefit cardiac, musculoskeletal, gastrointestinal and
immune systems in humans (17). Epidemiologic and preclinical findings also support their
anti-cancer properties. For example, n-3 FAs reduce onset of different cancers and protect
against late stage cancers in carcinogen induced mouse tumors, human tumor xenografts on
mouse, and spontaneous mouse tumors induced by transgenes. A higher intake of n-3
PUFAs is linked to a reduced risk of skin, colorectal, lung, prostate and breast cancers in
humans (18).
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The anticancer activities of EPA and DHA are partially associated with their effects on
modulating eicosanoid metabolism (19), including inhibition of PGE2 production. PGE2 is
derived from the n-6 Arachidonic acid (AA) precursors liberated from phospholipids in the
cell memberane and converted into PG by COX. Ample studies have implicated
proinflammatory and procarcerous effects of the inducible COX-2/PGE2 pathways in lung
cancer (20). We previously demonstrated that GSE might simultaneously function as a
natural COX-2 inhibitor and prostacyclin (PGI2) inducer. PGI2 is known for its
antineoplastic and anti-platelet properties, capable of improving endobronchial dysplasia in
former smokers (21). In this study, we demonstrate that oral LP significantly increases
serum PGE3, likely through the increase of precursor EPA. EPA can function as a selective
COX-2 inhibitor through competitive inhibition of the n-6 AA binding to COXs, resulting in
a decrease production of PGE2, while concomitantly generates PGE3. PGE3 has
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anitproliferative and anti-inflammatory activities, can potentially antagonize tumor

promoting effects of PGE2 in tumorigenic cells. Whereas the modulations of serum PGE3
significantly correlate with the modulations of Ki-67 LI, the modulations of EPA and DHA
only show a trend toward significant correlations with modulations of bronchial Ki-67 LI,
likely due to insufficient sample sizes.

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Since our participants were specifically instructed to not alter their dietary intake nor start
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new dietary supplements during the course of the study, we speculate that the increases in
DHA and EPA post-LP treatment likely are associated with the phytosome or lecithin
component of LP. Lecithin is comprised of mixtures of glycerophospholipids including PC
(22). it is conceivable that the phytosome increases systemic PC, which function as
transporters that enhance absorption and bioavailability of EPA and DHA from usual diet of
the participants. Such a notion is supported by a study showing that combined intake of
dietary crude lecithin with DHA increases systemic availability of DHA in rats (23). Long
chain n-3 PUFAs may also be synthesized from ALA by a progressive series of enzymatic
desaturation and chain elongation steps (24). To this end, significant increases in the
carnitine clusters of metabolites in the lipidomics likely reflect increases in carbon input
from phytosome and transport of long-chain fatty acids into mitochondria for subsequent β-
oxidation. The mechanisms involved in modulations of the carnitine cluster of metabolites
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and their potential anti-cancer roles in LP treatment remain to be elucidated. Significant

change in the plasmalogen PC(p-18:1/18:3) also indicates differences in peroxisome
metabolism, an organelle that is specifically active in PUFA metabolism that generates
plasmalogen precursors (25).

Our findings illustrate the novel, additive effects of phytosome in LP, beyond its original
intended purposes of enhancing absorption, intercellular transport and systemic
bioavailability of GSE. To our knowledge, this is the first report demonstrating the
additional, potential benefits of the carrier phytosome in humans. These findings, along with
the significant reduction of Ki-67 LI in the proximal bronchi, and favorable modulations of a
variety of other eicosanoids from our previous reports (7), further support the notion that
oral administration of LP is capable of dampening the driving forces of cancerizations
systemically and in the lungs (Fig. 2). Our findings support the continued clinical
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investigations of LP as an anti-neoplastic and chemopreventive agent against lung cancer.

Acknowledgement
We wish to thank A. Vargas and K. Park for their excellent technical assistance; Indena, Inc. and Thorne research,
Inc., for generously supplying the LP. This work has been supported by grants from the National Institute of Health:
(National Cancer Institute R21CA173211 to JTM), (NIH U2C ES030158 to OF); and VA Merit Review:
(BX002258, BX004092 and CX002028 to JTM). This research was partially supported by the University of New
Mexico Comprehensive Cancer Center Support Grant NCI P30CA118100. JTM is an Associate of the Kidney
Institute of New Mexico.

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Prevention Relevance Statement

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In this correlative study of leucoselect phytosome (LP) for lung cancer chemoprevention
in heavy active and former smokers, we demonstrate for the first time, favarble
modulations of omega-3 polyunsaturated fatty acid and downstream prostaglandin E3 in
fasting serum, further supporting the chemopreventive potential of LP against lung
cancer.
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Fig. 1.
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A). Impact Plot of modulations of Complex Lipidomics in fasting serum by LP treatement.

Oral LP treamtent significantly altered unsaturated FA, unsaturated PC, and carnitine
clusters. B). Three months of oral LP treatment significantly increased PGE3 levels in
fasting serum of study participants by an average of 26 % (66.2 + 10.3 pg/ml at baseline vs.
96.3 + 17.0 pg/ml at 3 months). Fold change of each biomarker from each participant was
calculated first by normalizing matched post-treatment to baseline pre-treatment values.
Columns, mean; bars, SEM (n = 6). *, P < 0.05. C). To determine the systemic,

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antineoplastic bioactivity of oral administration of LP, A549, H520, DMS114 and 1198 cells
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were treated with matched fasting heparinized plasma obtained from subjects pre- and
post-3 months of oral LP treatment (Plasma: culture medium = 1:10 for A549 and H520,
1:20 for DMS114 and 1198). Post-treatment plasma significantly reduced proliferation of
A549, H520, DMS114 and 1198 cells in comparison to pre-treatment plasma. Percent
change of cell viability from each participant was calculated first by normalizing matched
post-treatment to baseline pre-treatment values. Mean; bars, SEM (n = 6). ***, P < 0.001.
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Fig. 2.
Proposed mechanistic diagram of the effects of LP treatment on EPA, DHA, PC and major
eicosanoids against lung cancer. The phytosome portion of LP likely contains high levels of
PC and serves as transporters for n-3 PUFAs EPA and DHA. Increases in EPA and DHA
function as competitive inhibitors of COX-2. In addition, an increase in EPA results in an
increase in downstream PGE3. Other GSE-mediated modulations of major eicosanoids
signaling pathways previously described by our group are also depicted (a decrease in PGE2
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due to COX-2 inhibition by GSE, while an increase in prostacyclin synthase (PTGIS) by

GSE results in an overall increase in PGI2. Furthermore, an increase in 15-HETE is likely
due to shunting of the n-6 AA precursor toward the 15-LOX pathway in the setting of
COX-2 inhibition. Collectively, through modulations of these eicosanoids and n-3 PUFAs,
LP increases apoptosis, decreases cell proliferation and inflammation, thereby reduces the
driving forces of cancerization.

Cancer Prev Res (Phila). Author manuscript; available in PMC 2021 December 01.
 Mao et al. Page 14

Table 1A.

Systemic effects of 1 month of LP treatment on complex lipid metabolomics (n = 8)

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Cluster name Cluster size p-values FDR

Unsaturated FA 14 0.000014 0.00032

Unsaturated Phosphatidylcholines 75 0.000049 0.00038
Carnitine 6 0.000037 0.00038
Cholesterol Esters 9 1 1
Diglycerides 8 1 1
Galactosylceramides 6 1 1
Lactosylceramides 4 1 1
Lysophospholipids 3 1 1
NewCluster_14 6 1 1
NewCluster_18 5 1 1
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NewCluster_19 4 1 1
Phosphatidylethanolamines 16 1 1
Phospholipid Ethers 5 1 1
Plasmalogens 11 1 1
Saturated Ceramides 3 1 1
Saturated FA 7 1 1
Saturated_Lysophosphatidylcholines 8 1 1
Saturated_Phosphatidylcholines 7 1 1
Saturated Triglycerides 5 1 1
Sphingomyelins 23 1 1
Unsaturated Ceramides 12 1 1
Unsaturated Lysophosphatidylcholines 14 1 1
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Unsaturated_Triglycerides 51 1 1
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Cancer Prev Res (Phila). Author manuscript; available in PMC 2021 December 01.
 Mao et al. Page 15

Table 1B.

Key compounds in the unsaturated FA, the unsaturated PC clusters, and a plasmalogen that were significantly
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increased by LP treatment.

Compound Name Cluster name Pubchem. ID Fold change p-value

FA (20:5) (EPA) Unsaturated FA 446284 1.2 0.031

FA (22:6) (DHA) Unsaturated FA 445580 1.3 0.047
PC(39:6) Unsaturated_Phosphatidylcholines 52922637 1.3 0.031
PC(p-18:1/18:3) Plasmalogen 53480747 1.4 0.047
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Cancer Prev Res (Phila). Author manuscript; available in PMC 2021 December 01.
 Mao et al. Page 16

Table 1C.

Pearson corrrelation coefficients for modulations of PGE3, EPA, or DHA with modulations of bronchial Ki-67
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LI by LP (n = 6).

Ki-67 LI r value p value

PGE3 −0.900 0.037

EPA −0.794 0.109

DHA −0.709 0.178
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Cancer Prev Res (Phila). Author manuscript; available in PMC 2021 December 01.