Sorafenib increases cytochrome
P450 lipid metabolites in patient
with hepatocellular carcinoma
Can G. Leineweber
1
,
2
,
3
, Miriam Rabehl
1
,
2
, Anne Pietzner
1
,
2
,
Nadine Rohwer
1
,
2
,
4
, Michael Rothe
5
, Maciej Pech
6
, Bruno Sangro
7
,
Rohini Sharma
8
, Chris Verslype
9
, Bristi Basu
10
, Christian Sengel
11
,
Jens Ricke
12
, Nils Helge Schebb
13
, Karsten-H. Weylandt
1
,
2
*
†
and
Julia Benckert
14
*
†
1
Medical Department B, Division of Hepatology, Gastroenterology, Oncology, Hematology, Palliative
Care, Endocrinology, and Diabetes, Brandenburg Medical School, University Hospital Ruppin-
Brandenburg, Neuruppin, Germany,
2
Faculty of Health Sciences, Joint Faculty of the Brandenburg
University of Technology, Brandenburg Medical School and University of Potsdam, Potsdam, Germany,
3
Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain,
4
Department of
Molecular Toxicology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany,
5
Lipidomix, Berlin, Germany,
6
Department of Radiology and Nuclear Medicine, Otto-von-Guericke
University, Magdeburg, Germany,
7
Liver Unit and HPB Oncology Area, Clinica Universidad de Navarra and
CIBEREHD, Pamplona, Spain,
8
Department of Surgery and Cancer, Imperial College London, London,
United Kingdom,
9
Department of Digestive Oncology, University Hospitals Leuven, Leuven, Belgium,
10
Department of Oncology, University of Cambridge, Cambridge, United Kingdom,
11
Radiology
Department, Grenoble University Hospital, La Tronche, France,
12
Department of Radiology, University
Hospital, Ludwig-Maximilians-University (LMU) Munich, Munich, Germany,
13
Chair of Food Chemistry,
Faculty of Mathematics and Natural Science, University of Wuppertal, Wuppertal, Germany,
14
Department
of Hepatology and Gastroenterology, Charité—Universitätsmedizin Berlin, Corporate Member of Freie
Universität Berlin and Humboldt—Universität zu Berlin, Berlin, Germany
Hepatocellular carcinoma (HCC) is a leading cause of cancer death, and medical
treatment options are limited. The multikinase inhibitor sorafenib was the first
approved drug widely used for systemic therapy in advanced HCC. Sorafenib
might affect polyunsaturated fatty acids (PUFA)-derived epoxygenated metabolite
levels, as it is also a potent inhibitor of the soluble epoxide hydrolase (sEH), which
catalyzes the conversion of cytochrome-P450 (CYP)-derived epoxide metabolites
derived from PUFA, such as omega-6 arachidonic acid (AA) and omega-3
docosahexaenoic acid (DHA), into their corresponding dihydroxy metabolites.
Experimental studies with AA-derived epoxyeicosatrienoic acids (EETs) have
shown that they can promote tumor growth and metastasis, while DHA-
derived 19,20-epoxydocosapentaenoic acid (19,20-EDP) was shown to have
anti-tumor activity in mice. In this study, we found a significant increase in EET
levels in 43 HCC patients treated with sorafenib and a trend towards increased
levels of DHA-derived 19,20-EDP. We demonstrate that the effect of sorafenib on
CYP- metabolites led to an increase of 19,20-EDP and its dihydroxy metabolite,
whereas DHA plasma levels decreased under sorafenib treatment. These data
indicate that specific supplementation with DHA could be used to increase levels
of the epoxy compound 19,20-EDP with potential anti-tumor activity in HCC
patients receiving sorafenib therapy.
KEYWORDS
hepatocellular carcinoma, cytochrome P450, sorafenib, EET, EDP, omega-3 fatty acids,
oxylipins, lipidomics
OPEN ACCESS
EDITED BY
Annalisa Bruno,
University of Studies G. d’Annunzio Chieti
and Pescara, Italy
REVIEWED BY
Patrizia Ballerini,
University of Studies G. d’Annunzio Chieti
and Pescara, Italy
Stefania Tacconelli,
University of Studies G. d’Annunzio Chieti
and Pescara, Italy
*CORRESPONDENCE
Karsten-H. Weylandt,
karsten.weylandt@mhb-fontane.de
Julia Benckert,
julia.benckert@charite.de
†
These authors have contributed equally
to this work and share last authorship
SPECIALTY SECTION
This article was submitted to
Inflammation Pharmacology,
a section of the journal
Frontiers in Pharmacology
RECEIVED 14 December 2022
ACCEPTED 15 February 2023
PUBLISHED 03 March 2023
CITATION
Leineweber CG, Rabehl M, Pietzner A,
Rohwer N, Rothe M, Pech M, Sangro B,
Sharma R, Verslype C, Basu B, Sengel C,
Ricke J, Schebb NH, Weylandt K-H and
Benckert J (2023), Sorafenib increases
cytochrome P450 lipid metabolites in
patient with hepatocellular carcinoma.
Front. Pharmacol. 14:1124214.
doi: 10.3389/fphar.2023.1124214
COPYRIGHT
© 2023 Leineweber, Rabehl, Pietzner,
Rohwer, Rothe, Pech, Sangro, Sharma,
Verslype, Basu, Sengel, Ricke, Schebb,
Weylandt and Benckert. This is an open-
access article distributed under the terms
of the Creative Commons Attribution
License (CC BY). The use, distribution or
reproduction in other forums is
permitted, provided the original author(s)
and the copyright owner(s) are credited
and that the original publication in this
journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms.
Frontiers in Pharmacology frontiersin.org01
TYPE Original Research
PUBLISHED 03 March 2023
DOI 10.3389/fphar.2023.1124214
1 Introduction
Liver cancer is a global issue, being the most common cancer
and the leading cause of cancer death in transition countries. In
2020, almost 9,06,000 patients were diagnosed with liver cancer and
over 8,30,000 deaths were documented worldwide. Hepatocellular
carcinoma (HCC) has the highest prevalence among the different
subtypes of liver cancer (Sung et al., 2021). Viral infections, more
specifically hepatitis B and C virus (Datfar et al., 2021), lifestyle
factors such as alcohol intake (Matsushita and Takaki, 2019), as well
as type 2 diabetes and non-alcoholic fatty liver disease (NAFLD)
(Estes et al., 2018) remain the leading risk factors, depending on the
region considered. Hepatocellular carcinoma remains one of the
most common causes of cancer death, especially in men, and has one
of the lowest 5-year survival rates of all different cancer types (Siegel
et al., 2022).
In addition to chronic inflammation, tissue remodeling and
changes in cellular signaling are pathogenetic factors of
carcinogenesis (Refolo et al., 2020). Interestingly, patients with a
non-cirrhotic HCC, mostly caused by NAFLD, seem to show a more
severe HCC histopathology on one hand but better overall survival
on the other hand (Gawrieh et al., 2019).
Limited stages of HCC can be treated with locoregional
procedures such as surgical (e.g., resection, transplantation) or
radiological (e.g., transarterial chemoembolization, selective
internal radiotherapy) intervention, prolonging survival for more
than 5 years, depending on the underlying liver disease. Once the
disease progresses, survival is compromised to approximately 1 year
even with systemic therapy (Forner et al., 2018;Vogel et al., 2022).
Systemic therapy options were shown to increase overall survival
(OS) and progression-free survival (PFS). However, either applying
combination therapies including immune checkpoint inhibitors
such as atezolizumab plus bevacizumab (OS 13,4-19,2 months;
PFS 6,9 months) (Finn et al., 2020) or tyrosine kinase inhibitors
lenvatinib (OS 13,6 months; PFS 7,3 months) (Kudo et al., 2018)or
sorafenib (OS 10,7-15,5 months; PFS 3,6-5,5) (Kelley et al., 2022) has
not altered the severity or mortality of the disease so far (Vogel et al.,
2022).
The first targeted and currently a widely used systemic therapy
for HCC is the oral multikinase inhibitor sorafenib binding in an
ATP-binding pocket to inhibit kinase function (Hwang et al., 2013),
which predominantly inhibits angiogenesis via binding the vascular
endothelial growth receptor (VEGFR). Furthermore, it targets the
cell proliferation and differentiation via the rapidly accelerated
fibrosarcoma (RAF) signaling pathway (Wilhelm et al., 2004) and
the platelet-derived growth factor receptor-β(PDGFR-β)(Mody
and Abou-Alfa, 2019) as well as the beneficial effects, particularly in
cancer and its complications, which is likely due to the inhibition of
nuclear factor kappa B (NF-κB) and production of the pro-
resolution mediators at the molecular level (Freitas and Campos,
2019).
In addition to the known anti-angiogenic and anti-proliferative
effects of sorafenib it has also been described to have effects on the
soluble epoxide hydrolase (sEH) which showed similar anti-
inflammatory effects as conventional sEH inhibitors in
lipopolysaccharide-induced inflammation models in mice (Liu
et al., 2009). Sorafenib is a potent inhibitor of sEH compared
with conventional urea-based sEH inhibitors (Hwang et al.,
2013). The sEH is expressed in numerous human tissues with the
main distinction between microsomal epoxide hydrolase (mEH) and
sEH (Morisseau and Hammock, 2013). The sEH metabolization is
the dominant pathway in humans, so that it can be assumed that
sEH inhibition has a stabilizing effect on endogenous epoxy
metabolites in tissues (Spector and Kim, 2015).
The epoxidation of long-chain polyunsaturated fatty acids (LC-
PUFAs), such as the omega-6 (n-6) PUFA arachidonic acid (C20:
4n6, AA), as well as the omega-3 (n-3) PUFAs eicosapentaenoic acid
(C20:5n3, EPA) and docosahexaenoic acid (C22:6n3, DHA) to
epoxyeicosatrienoic acids (EETs), epoxyeicosatetraenoic acids
(EEQs) and epoxydocosapentaenoic acids (EDPs), respectively,
are catalyzed by the cytochrome P450 (CYP) epoxygenases
(Figure 1). These epoxymetabolites are then further metabolized
via she into their biologically less active corresponding dihydroxy
metabolites (Zhang et al., 2014;Wang et al., 2018).
The role of epoxidized LC-PUFAs is well established in several
biological processes, such as angiogenesis, inflammation, and tumor
growth: In different animal models, it has been shown that sEH-
inhibition has a positive influence on cardiovascular and liver
abnormalities (Iyer et al., 2012), liver fibrosis and portal
hypertension (Zhang et al., 2018), fatty liver (Yao et al., 2019)
and non-alcoholic steatohepatitis (Wang et al., 2019). The EETs
are known to affect blood pressure, inflammation, pain sensation,
and regeneration (Arnold et al., 2010;Morisseau et al., 2014).
However, a proangiogenic effect of 11,12-EET and 14,15-EET,
the main EET regioisomers in mammals (Spector and Kim,
2015), has been described via the epidermal growth factor (EGF)
and VEGF pathways (Zhang et al., 2014), which may explain the
finding that EETs promote tumor growth. The n-6 AA-derived
regioisomers 5,6-EET and 8,9-EET were found to increase cell
proliferation and de novo vascularization (Yan et al., 2008),
whereas 11,12-EET and 14,15-EET promote tumor angiogenesis
through endothelial cell proliferation (Panigrahy et al., 2012;Zhang
et al., 2013). An increase in 14,15-EET through sEH-inhibition led to
increased tumor growth and metastasis through cell invasion in
experimental studies (Panigrahy et al., 2012). In summary, through
mechanisms of cell proliferation, de novo vascularization and
endothelial proliferation several EET-regioisomers promote
tumor growth and metastasis (Yan et al., 2008;Panigrahy et al.,
2012;Zhang et al., 2013;Zhang et al., 2014). The sEH-inhibitory
effect of sorafenib might thus carry clinically relevant consequences
by increasing these pro-tumorigenic EET mediators.
In contrast, n-3 PUFA-derived regioisomers show anti-
angiogenic effects both via the VEGF and FGF-2 pathway
(Zhang et al., 2014). In a tumor mouse model, it was shown,
that low dose sEH-inhibition led to an increase in n-3 DHA-
derived 19,20-EDP and thereby reduced tumor angiogenesis and
cell invasion and thus inhibition of tumor growth (Zhang et al.,
2013). Furthermore, a protective effect in obesity and obesity-
related comorbidities, such as fatty liver disease, of n-3 epoxy
PUFA was found in animal models (López-Vicario et al., 2015).
The beneficial effects of n-3 PUFA regarding to cancer and its
complications are probably due to their anti-inflammatory and
pro-resolution mediators (Freitas and Campos, 2019). In the
context of abundant DHA, the sEH-inhibitory effect of sorafenib
mightthusleadtohigherlevelsof19,20-EDP,mediatinganti-
tumor effects.
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Leineweber et al. 10.3389/fphar.2023.1124214
Currently, n-6 PUFA are found in a ratio of approximately
20 times more than n-3 PUFA in the human diet (Harris, 2006);
therefore humans have low n-3 PUFA tissue levels, and a shift of the
competitive n-6 and n-3 PUFA metabolism towards n-6 PUFA
derived lipid metabolites (Spector and Kim, 2015).
We therefore aimed to investigate levels of n-6 and n-3 PUFA in
HCC patients, as well as n-6 PUFA- and n-3 PUFA-derived epoxide
and corresponding dihydroxy compounds in HCC patients without
and during sorafenib therapy. Based on the data of our pilot study
(Leineweber et al., 2020), we hypothesized that sorafenib treatment,
due to sEH-inhibitory and possibly CYP-modulating effects, might
increase the presence of potentially tumor growth-suppressing
DHA-derived EDPs, as well as of potentially tumor growth-
promoting EETs.
2 Materials and methods
2.1 Patients and blood sampling
The study population evaluated in this sub-analysis comprised
patients within the palliative treatment arm of the randomized,
controlled, multicenter phase II SORAMIC study, which evaluated
sorafenib alone compared to selective internal radiation therapy
(SIRT) combined with sorafenib on overall and progression-free
survival in patients with advanced HCC (Ricke et al., 2019).
Patients were included in this analysis if they received study
treatment in the palliative arm of SORAMIC and signed an
informed consent form, so blood samples were collected and
analyzed at baseline (BL) and at the first follow-up visit (FU) after
approximately 7–9 weeks, and samples were stored for
subsequent analyses at −80°C. Of the 424 randomized patients
assignedtothepalliativearm,weperformed lipidomic analysis of
43 patients from the intention to treat (ITT) population, all of
whom received sorafenib, characterized in terms of gender
distribution, age, body mass index (BMI), presence of
cirrhosis, liver function, biomarker, or tumor stage according
to Barcelona Clinic Liver Cancer (BCLC) stage, except in the
expression of the Child-Pugh points between 5 and 6/7 (p<
0.0453) as shown in Table 1.
2.2 Sample preparation and GC
Plasma samples were analyzed for determination of fatty acids
using the gas chromatography (GC) technology as described previously
(Wang et al., 2022). 75 μL of EDTA plasma per sample was used for the
GC preparation. Methylation and extraction of FAs were carried out on
the basis of an established protocol (Kang and Wang, 2005). Briefly,
frozen samples were thawed at room temperature. All samples were
then mixed with 50 μL pentadecanoic acid (PDA, 1 mg/mL in ethanol,
Merck Schuchardt OHG, Hohenbrunn, Germany) as internal standard,
500 μLborontrifluoride (BF
3
, Sigma-Aldrich Chemie GmbH,
Taufkirchen, Germany) in 14% methanol (Merck KGaA, Darmstadt,
Germany), and 500 μLn-hexane (Merck KGaA, Darmstadt, Germany)
in glass vials and were tightly closed. After vortexing, samples were
incubated for 60 min in a preheated block at 100°C. After cooling down
to room temperature, the mixture was added to 750 μLwater,vortexed,
and extracted for 4 min. Then all samples were centrifuged for 5 min
(RT, 3,500 rpm). From each sample, 100 μL of the upper n-hexane layer
was transferred into a micro-insert (placed in a GC glass vial), tightly
closed, and analyzed by GC.
GC was performed on a 7890B GC System (Agilent Technologies,
Santa Clara, CA, United States) withanHP88Column(112/8867,
60 m × 0.25 mm × 0.2 μm, Agilent Technologies, Santa Clara, CA,
United States), with the following temperature gradient: 50°C–150°C
with 20°C/min, 150°C–240°Cwith6
°C/min, and 240°C for 10 min (total
run time 30 min). Nitrogen was used as carrier gas (constant flow 1 mL/
min). 1 μL of each sample was injected into the injector (splitless
injection, 280°C). The flame ionization detector (FID) analysis was
performed at 250°C with the following gas flows: hydrogen 20 mL/min,
air 400 mL/min, and make up (nitrogen) 25 mL/min. Methylated FAs
in the samples were identified by comparing the retention times with
thoseofknownmethylatedFAsoftheSupelco
®
37 FAME MIX
standard (CRM47885, Sigma Aldrich, Laramie, WY, United States)
and a mix of single FAME standards [DPA, C22:5 n-3, AdA, C22:4 n-6
(Cayman Chemicals, Ann Arbor, MI, United States)]. Analysis and
integration of the peaks were carried out with OpenLAB CDS
ChemStation Edition (Agilent Technologies, Santa Clara, CA,
United States). FA values are presented as percentage (Ramsay et al.,
2018) of total FA content and absolute concentrations (µg/mL). For the
study, 16 FAs were included as follows: myristic acid (C14:0), palmitic
FIGURE 1
CYP-dependent lipid metabolite formation from AA/DHA/EPA, and the potential effects of sorafenib.
Frontiers in Pharmacology frontiersin.org03
Leineweber et al. 10.3389/fphar.2023.1124214
acid (C16:0), stearic acid (C18:0), arachidic acid (C20:0), behenic acid
(C22:0), lignoceric acid (C24:0), palmitoleic acid (C16:1 n-7c), oleic acid
(C18:1 n-9c), nervonic acid (C24:1 n-9), eicosapentaenoic acid (EPA,
C20:5 n-3), docosapentaenoic acid (DPA, C22:5 n-3), docosahexaenoic
acid (DHA, C22:6 n-3), linoleic acid (LA, C18: 2 n-6), dihomo-gamma-
linolenic acid (DGLA, C20:3 n-6), arachidonic acid (AA, 20:4 n-6), and
adrenic acid (AdA, C22:4 n-6).
2.3 Sample preparation and LC/ESI-MS/MS
Plasma samples were analyzed for epoxymetabolites using the LC/
ESI-MS/MS lipidomics technology as described previously (Fischer
et al., 2014). Lipid mediators and deuterated standards used in this
study were purchased from Cayman Chemical (Ann Arbor, MI,
United States). Materials used for solid phase extraction (SPE), such
as sodium acetate, ethyl acetate, acetic acid, and n-hexane were obtained
from Fisher Scientific (Loughborough, UK). Additionally, 99%
butylated hydroxytoluene (BHT, 2,6-di-tert-butyl-4-methylphenol)
was obtained from Acros Organics (Geel, Belgium), and Bond Elute
Certify II columns from Agilent Technologies (Santa Clara, CA,
United States) were used. LC-MS solvents, such as methanol
(Lichrosoly hypergrade) and acetonitrile (Lichrosoly hypergrade),
were obtained from Merck (Darmstadt, Germany).
For sample preparation, an internal standard consisting of 14,15-
DHET-d11, 15-HETE-d8, 20-HETE-d6, 8,9-EET-d11, 9,10-DiHOME-
d4, 12,13-EpOME-d4, 13-HODE-d4 and LTB4-d4 (500 pg each) and
ice-cold methanol containing BHT (0.1%) was added to 200 mL
plasma. After alkaline hydrolysis using 1 mmol sodium hydroxide
the pH was adjusted with acetic acid and sodium acetate buffer
containing 5% v/v methanol at pH 6. After centrifugation, the
obtained supernatant was added to SPE columns, which were
preconditioned with 3 mL methanol, followed by 3 mL of 0.1 mol/L
sodium acetate buffer containing 5% methanol (pH 6). The SPE
columns were then washed with 3 mL methanol/H2O (50/50, v/v).
For elution, 2.0 mL of n-hexane:ethyl acetate (25:75) with 1% acetic acid
were used. The extraction was performed with a SUPELCO Visiprep
manifold. The eluate was evaporated on a heating block at 40°Cundera
stream of nitrogen. The solid residue was resolved in 100 µL 60%
methanol in water.
The prepared samples were analyzed using an Agilent 1290 HPLC
system with a binary pump, an autosampler, and a column thermostat
with a Agilent Zorbax Eclipse plus C18 column 150 mm × 2.1 mm,
1.8 µm using a solvent system of aqueous acetic acid (0.05%) and
acetonitrile:methanol (50:50). The multiple step elution gradient started
at 95% aqueous phase, which was increased within 18 min–98% organic
phaseandheldtherefor10min.Theflow rate was set at 0.3 mL/min,
and injection volume was 20 μL. The HPLC was coupled with an
Agilent 6495 Triple Quad mass spectrometer with an electrospray
ionization source. Analysis of lipid mediators was performed with the
Multiple Reaction Monitoring in the negative mode, limit of
quantitation (LOQ) was 0.01 ng/mL.
2.4 Statistical analysis
Statistical analysis was performed using GraphPad Prism 9
(GraphPad Software, La Jolla, CA, United States). The
comparison was made using the Wilcoxon matched-pairs signed-
rank test. The correlation was made using linear regression. All
values are presented as the mean ± standard error of the mean.
Statistical significance was assumed when p<0.05. (*0.01 ≤p<0.05;
**0.001 ≤p<0.01; ***p<0.001).
3 Results
Blood samples from a total of n= 43 HCC patients were analyzed in
a paired fashion with blood taken without and undergoing 7–9 weeks
sorafenib treatment. This is a sub-analysis of the well-characterized
study population of the randomized controlled, multicenter phase II
TABLE 1 Patient characteristics of the n= 43 HCC patients receiving sorafenib
treatment. Data are presented as mean ± standard error of the mean.
Characteristic Total (n= 43)
Gender
Female 5 (12%)
Male 38 (88%)
Age (years) 66.97 ± 2.19
BMI 26.98 ± 0.99
Liver disease
Alcohol 17 (40%)
Hepatitis B/C 15 (35%)
Liver cirrhosis 36 (84%)
Child Pugh
5 28 (65%)
6/7 15 (35%)
BCLC
B 16 (37%)
C 27 (63%)
Further disease classification
Liver dominant disease 41 (95%)
Portal vein invasion 16 (37%)
Extrahepatic metastases 8 (19%)
Bilirubin (µmol/L) at baseline 15.91 ± 1.66
Albumin (g/L) at baseline 37.68 ± 1.42
AlBi Score at baseline −2.44 ± 0.13
DeRitis quotient 1.65 ± 0.26
AFP
<400 ng/mL 25 (58%)
>400 ng/mL 16 (37%)
Sorafenib: Daily dose (mg) 410.82 ± 23.40
Overall survival (months) 12.04 ± 1.46
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Leineweber et al. 10.3389/fphar.2023.1124214
SORAMIC trial (Ricke et al., 2019). Patients from this study population,
from which suitable amounts of blood samples were available for the
analysis of fatty acids and their lipid metabolites before and during
sorafenib treatment, were chosen for the analysis performed and
presented here. The patients received sorafenib 200 mg twice a day
for 1 week before increasing the dose to 400 mg twice a day. Based on
disease progression and clinical condition, the sorafenib dose was
escalated to 600–800 mg or reduced to 0–200 mg. The patients’
general characteristics are shown in Table 1.
3.1 N-6 and n-3 epoxides and dihydroxy
metabolites are higher in patients
undergoing sorafenib treatment
To investigate the effect of sorafenib treatment on the n-6 and n-
3 PUFA epoxide formation, we measured the concentrations of the
epoxymetabolites and corresponding dihydroxy metabolites by
quantitative LC-ESI-MS/MS analysis in plasma samples of HCC
patients without and during sorafenib therapy. As a result of
sorafenib treatment, the levels of n-6 AA-derived
epoxymetabolites 5,6-EET and 8,9-EET increased significantly.
Levels of epoxymetabolites derived from the n-3 PUFAs tended
to increase as well, but for DHA- and EPA-derived epoxides failed to
reach significance (Figures 2A–C;Supplementary Figures S1A–C).
The concentrations of EETs were higher compared to the n-3
PUFA-derived EEQs. EDP metabolite concentrations were
approximately half of those observed for the EETs, while the
concentrations of the EEQs were the lowest in this patient cohort.
The dihydroxy-PUFA products of the epoxy-PUFA formed via
the sEH, respectively dihydroxyeicosatrienoic acids (DHETs) from
EETs, dihydroxydocosapentaenoic acids (DiHDPAs) from EDPs
and dihydroxyeicosatetraenoic acids (DiHETEs) from EEQs
increased whilst on sorafenib treatment as well (Supplementary
Table S1). When comparing absolute amounts of AA-, DHA- and
EPA-derived epoxy-plus dihydroxy-PUFA significantly higher
levels of metabolites derived from all three PUFAs were found
(Figures 2D, E).
3.2 N-6 and n-3 fatty acid levels decrease
during sorafenib treatment
To explore the presence of LC-PUFA, the fatty acid composition
in plasma from patients with HCC was analyzed without and during
sorafenib treatment by gas chromatography.
FIGURE 2
Effects on the concentrations of (A) AA-, (B) DHA-, and (C) EPA-derived epoxy-PUFA EETs, EDPs, and EEQs; and (D) AA-derived epoxy-PUFA plus
dihydroxy-PUFA, (E) DHA-derived epoxy-PUFA plus dihydroxy-PUFA, and (F) EPA-derived epoxy-PUFA plus dihydroxy-PUFA in the plasma of n=
43 patients with hepatocellular carcinoma (HCC) without and undergoing sorafenib treatment (ng/mL ± standard error of the mean). Statistical
differences were determined using the Wilcoxon signed-rank test (**p<0.01; ***p<0.001; ****p<0.0001).
Frontiers in Pharmacology frontiersin.org05
Leineweber et al. 10.3389/fphar.2023.1124214
Interestingly, we found a decrease in the relative content of n-6
(AA) and n-3 (DHA) PUFAs, with an increasing n-6/n-3 ratio
(Figure 3A;Supplementary Table S2).
HCC patients without sorafenib had significantly higher levels of
monounsaturated FAs (MUFA, p<0.001)—comprising palmitoleic
acid (C16:1 n-7c), oleic acid (C18:1 n-9c), and nervonic acid (C24:
1 n-9)—and significantly lower levels of PUFAs (p<0.01)—
comprising EPA (C20:5 n-3), docosapentaenoic acid (DPA, C22:
5 n-3), DHA (C22:6 n-3), linoleic acid (LA, C18: 2 n-6), dihomo-
gamma-linolenic acid (DGLA, 20:3 n-6), AA (20:4 n-6), and adrenic
acid (AdA, C22:4 n-6)—but there was no significant difference in the
content of saturated fatty acids (SFA)—comprising myristic acid
(C14:0), palmitic acid (C16:0), stearic acid (C18:0), arachidic acid
(C20:0), behenic acid (C22:0), and lignoceric acid (C24:
0)—(Figure 3B). With respect to specific fatty acids, HCC
patients whilst on sorafenib treatment had a significantly lower
relative content of the n-3 PUFA DHA (p<0.0001), thus a
significant decrease in DHA + EPA (p<0.05), analogous to the
HS-Omega-3 Index (von Schacky, 2020), and also a significant lower
relative content of the n-6 PUFA AA (p<0.0001).
3.3 N-6 and n-3 cytochrome P450 epoxy
and dihydroxy product ratios do not support
the hypothesis of increased sEH inhibition
during sorafenib treatment
To determine whether the sEH inhibitory effect of sorafenib is
detectable from the lipid metabolites assessed here, we analyzed whether
sorafenib treatment increases plasma content of epoxymetabolites as
compared to their dihydroxy products. As a marker for the enzyme
activity in the CYP epoxygenase/sEH axis the plasma ratio of EET to
DHET as characterization of the sEH inhibition has been used (Liu
et al., 2009). We adapted this approach using the following equation for
the AA-as well as the DHA- and EPA derived epoxy- and dihydroxy
compounds:
sEH −Activity dihydroxy −PU FA
epoxy −PUFA
However, in contrast to lower ratios that would indicate lower
sEH activity, we found higher dihydroxy/epoxy product ratios in the
sorafenib treated patients (Figure 4). This does not support lower
sEH activity in the sorafenib-treated patients. Interestingly, these
higher ratios were only significant for the n-3 PUFA DHA- and
EPA-derived metabolites.
3.4 Metabolization of AA and EPA to their
derived cytochrome P450 epoxy and
dihydroxy products is similar, while
metabolization of DHA to 19,20-EDP and
19,20-DiHDPA is markedly higher
In order to assess total CYP-epoxide and corresponding
dihydroxy formation as a function of their respective substrate
fatty acids we analyzed the epoxy and corresponding dihydroxy
concentrations as a ratio with their respective substrate PUFA:
CYP −products epoxy +dihydroxy PU FAs
PUFAs
We found higher CYP product/PUFA ratios due to sorafenib
treatment, providing evidence of increased presence of bioactive
epoxy-PUFA from AA, EPA, and DHA in patients undergoing
sorafenib treatment (Figures 5A–C). Furthermore, when analyzed as
a ratio of DHA-derived CYP-products versus DHA as substrate, the
19,20-metabolites were found to be the predominant metabolites
formed (Figure 5B).
FIGURE 3
(A) Relative n-3 (docosahexaenoic acid, DHA; eicosapentaenoic acid, EPA) and n-6 (arachidonic acid, AA) PUFA levels in plasma from n= 43 patients
with hepatocellular carcinoma (HCC) without and during sorafenib treatment individually, summarized and as a ratio. (B) Relative content of saturated
fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) in plasma from n= 43 patients with HCC without and
undergoing sorafenib treatment. Statistical differences were determined using the Wilcoxon signed-rank test (*p<0.05, **p<0.01, ***p<0.001,
****p<0.0001).
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Leineweber et al. 10.3389/fphar.2023.1124214
FIGURE 4
Ratio of n-6 and n-3 PUFA-derived dihydroxy to epoxy-PUFA as a marker for sEH activity in n= 43 patients with HCC without and undergoing
sorafenib treatment (***p<0.001).
FIGURE 5
N-3 and n-6 PUFA-derived epoxides plus dihydroxy compounds as a marker for the presence of CYP metabolites in plasma from n= 43 patients with
HCC without and undergoing sorafenib treatment. (A) Ratio of AA-derived products divided by AA plasma content, (B) ratio of DHA-derived products
divided by DHA plasma content, (C) ratio of EPA-derived products divided by EPA plasma content (*p<0.05, **p<0.01; ***p<0.001; ****p<0.0001).
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Leineweber et al. 10.3389/fphar.2023.1124214
4 Discussion
We found significant differences in plasma fatty acid
composition in patients with HCC without sorafenib compared
to during sorafenib treatment. Relative levels of AA and DHA were
significantly lower during sorafenib treatment. Furthermore, we
were able to demonstrate significantly higher EET levels and a
trend towards increased n-3 CYP metabolites especially 19,20-
EDP in this study population with HCC receiving sorafenib
treatment.
When taking into account the different levels of the precursor n-
3 PUFAs EPA and DHA we were able to establish that EPA is
metabolized by CYP enzymes to a similar extent as AA, while DHA
utilization was higher, leading to significantly increased levels of the
19,20-metabolites derived from DHA during sorafenib treatment
(Figures 2E,5B). Given that previous data from mouse models show
inhibition of tumor angiogenesis and reduced cell invasion by
increasing 19,20-EDP (Zhang et al., 2013) and to dampen and
alleviate inflammation in the liver (López-Vicario et al., 2015),
this could be a beneficial effect of sorafenib that could be
harnessed in HCC therapy by supplementing DHA.
Generally, a beneficial role of n-3 PUFAs to dampen
development of HCC is described both in animal models (Lim
et al., 2009;Weylandt et al., 2011;Inoue-Yamauchi et al., 2017) and
human observation studies (Sawada et al., 2012;Gao et al., 2015;
Koh et al., 2016). This and the disbalance of the n-6/n-3 ratio in the
Western Diet (Harris, 2006) suggests that supplementation of n-3
PUFAs could balance the n-6/n-3 ratio and may reduce tumor
progression in HCC patients, regardless of sorafenib treatment.
With all the described effects of n-3 PUFAs as receptor agonists,
modulators of molecular signalling pathways and inflammatory
responses, and data indicating that n-3 PUFA increase the
efficacy of chemotherapies and consequently the overall survival
of cancer patients, n-3 PUFAs can thus be considered as
pharmaceutical nutrients (Bougnoux et al., 2009;Chagas et al.,
2017;Paixão et al., 2017). However, in the population studied
here the n-3 PUFA baseline was not associated with the overall
survival (Supplementary Figure S2).
Prior studies showed that dietary increase of baseline n-3
PUFA concentrations can enhance formation of n-3 PUFA-
derived CYP epoxy-PUFA (Fischer et al., 2014;Sarparast
et al., 2020;Weylandt et al., 2022). Higher levels of n-3 PUFA
may thus potentially increase anti-tumor n-3 PUFA-derived
epoxymetabolites as well as decrease pro-tumor n-6 PUFA-
derived metabolites (Zhang et al., 2014). Interestingly, in this
study we found lower levels of DHA in patients treated with
sorafenib (Figure 3), further supporting the concept to increase
DHAinthedailydietinordertoincreasealsolevelsof19,20-
EDP in HCC patients treated with sorafenib.
Many classes of currently used drugs can block or modify
pathways of lipid mediator formation. Particularly well-
established are non-steroidal anti-inflammatory drugs inhibiting
the cyclooxygenase (COX) enzymes as well as numerous
clinically well-established substances that modify (induce, inhibit)
CYP enzymes and thereby modify lipid mediator formation. In
general, by using quantitative LC-MS/MS oxylipin analysis in the
context of established pharmacotherapy (pharmacolipidomics) as
shown in this paper we hope to identify oxylipins that might be used
to stratify and possibly also modify and improve response to
treatments: Experimental data indicate strong biological effects of
specific lipid mediators, particularly with regard to inflammation-
dampening oxylipins from n-3 PUFA (n-3 IDOs) (Weylandt et al.,
2022) in contrast to often inflammation-promoting oxylipins from
n-6 PUFA (n-6 IPOs). As concentrations of these can be modified by
changes in fatty acids substrates, as well as established drugs such as
sorafenib, there could be a rationale for targeted modifications of the
n-6/n-3 PUFA ratio in the diet in the context of established
pharmacotherapy to harness these effects.
In systemic HCC therapy, the combination of immune
checkpoint inhibitors, and VEGF pathway inhibitors such as
sorafenib could promote an immune-permissive environment,
thereby enhancing the response to immune therapeutic
approaches. Immunotherapy is effective in only approximately 1/
3 of cancer patients, and targeting the TME to decrease tumor cell
evasion is regarded as an opportunity to improve response to
immunotherapy, as conversion of “cold”tumors to “hot”tumors
with T cell infiltration is associated with a better response rate to
cancer treatment (Bonaventura et al., 2019). TME-modifying
properties of lipid mediator levels may therefore enhance
antitumor effects by transforming the immune landscape. Aspirin
dampens tissue inflammation via the metabolism of arachidonic
acid in the COX enzymatic pathways and can reduce cellular growth
in hepatocellular carcinoma (Tao et al., 2018;Refolo et al., 2020).
Whether a CYP/sEH-dependent effect on lipid mediators—which
could be modulated/enhanced by sorafenib as described
here—could also play a role in the TME, remains a topic for
future studies that are directly analyzing effects of n-3 IDO and
n-6 IPO levels and formation, as well as immune cells in liver and
liver tumor tissue.
In our data presented here, we were not able to discern an sEH
inhibitory effect of sorafenib (Figure 4). Non-etheless we established
significantly higher levels of CYP-derived epoxy and dihydroxy
metabolites in patients undergoing sorafenib treatment. While
previous results from one of us show that storage at −80°Cshould
be sufficient to yield stable CYP derived oxylipin readings comparable
to other oxylipins (Gladine et al., 2019;Koch et al., 2020), there might
have been variations in the process of blood sampling and storage
leading to changes in epoxy-PUFA levels. Another explanation would
be that sorafenib may have more complex effects on PUFA-derived
metabolites in humans, with increased formation of epoxy-PUFAs. We
did not analyse expression of sEH directly, therefore there is a possibility
of increased sEH expression, as described in an animal models of high
fat diet induced liver disease (López-Vicario et al., 2015)whichmight
compensate for an inhibitory effect of sorafenib. Interestingly, in our
pilot study we found an increase of 8,9-EET, 11,12-EET and 14,15-EET
levels in HCC patients treated with sorafenib (Leineweber et al., 2020)
while we found a significant increase only of 5,6-EET and 8,9-EET levels
here. We believe this might be due to the analytical limitations due to
differences in sample taking and storage, and not a mechanistic
difference in the effect observed. Indeed, we suggest to use the
combined analysis of epoxides and dihydroxy compounds (as in
Figures 2D–F) to assess epoxy metabolite formation. However future
studies could better address this question by analyzing the samples
under defined conditions and at different time intervals after blood
drawing to measure the effects of these variations on epoxy- and
dihydroxy-PUFA levels.
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Leineweber et al. 10.3389/fphar.2023.1124214
5 Conclusion
In this study, we investigated the effect of sorafenib treatment on
PUFA formation and epoxy lipid mediator concentrations in
peripheral blood plasma in a group of 43 HCC patients as a sub-
analysis of the randomized, controlled, multicenter phase II
SORAMIC study. We were able to demonstrate markedly
increased epoxy plus dihydroxy PUFA concentrations in the
peripheral blood of HCC patients undergoing sorafenib therapy.
These results support previous findings that sorafenib treatment
induces a change in epoxy-/dihydroxy-PUFA concentrations.
Given the anti-tumor effects described in experimental models for the
n-3 PUFA-derived 19,20-EDP, these data further support the hypothesis
that dietary n-3 PUFA supplementation in addition to sorafenib
treatment could contribute anti-tumor effects due to n-3 epoxy-PUFA.
Data availability statement
The raw data supporting the conclusion of this article will be
made available by the authors, without undue reservation.
Ethics statement
The studies involving human participants were reviewed and
approved by Ethics committee Otto-von-Guericke University
Magdeburg, Germany and Local Ethics committees. The patients/
participants provided their written informed consent to participate in
this study.
Author contributions
Conceptualization: CL, JR, NS, JB, and K-HW; Methodology: JR,
JB, CL, NS, MRo, NR, AP, and K-HW; Analysis: CL, MRa, AP, NR,
NS, MRo, and K-HW; Investigation: CL, MRa, AP, and K-HW;
Resources: JR, JB, CL, AP, and MRo; Data curation: JR, JB, MP, BS,
RS, CV, BB, CS, CL, and MRa; Writing—original draft preparation:
CL; Writing—review and editing: JR, NS, JB, and K-HW;
Visualization: CL, MRa, and K-HW; Supervision: JB and K-HW;
Project administration: JR and K-HW. All authors have read and
agreed to the published version of the manuscript.
Acknowledgments
We thank the SORAMIC research group for providing the
samples, recruiting patients, collecting patient information, and
processing blood samples, and the patients for their
participation. This research was supported by the NIHR
Cambridge Biomedical Research Centre (BRC-1215-20014).
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fphar.2023.1124214/
full#supplementary-material
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Glossary
AA arachidonic acid
AFP alpha-fetoprotein
AlBi albumin-bilirubin
BCLC barcelona clinic liver cancer
BHT butylated hydroxytoluene
BMI body mass index
COX cyclooxygenase
CT computer tomography
CYP cytochrome P450
DHA docosahexaenoic acid
DHET dihydroxyeicosatrienoic acid
DiHDPA dihydroxydocosapentaenoic acid
DiHETE dihydroxyeicosatetraenoic acid
EDP epoxydocosapentaenoic acid
EEQ epoxyeicosatetraenoic acid
EET epoxyeicosatrienoic acid
EGF epidermal growth factor
EPA eicosapentaenoic acid
FGF fibroblast growth factor
FID flame ionization detector
GC gas chromatography
HCC hepatocellular carcinoma
HPLC/ESI high performance liquid chromatography electrospray
ionization
LC-PUFA long-chain polyunsaturated fatty acid
mEH microsomal epoxide hydrolase
MRI magnetic resonance imaging
MS mass spectrometry
NAFLD fatty acid non-alcoholic fatty liver disease
NASH non-alcoholic steatohepatitis
NF-κBnuclear factor kappa B
n-3 PUFA omega-3 polyunsaturated fatty acids
n-6 PUFA omega-6 polyunsaturated fatty acids
OS overall survival
PDA pentadecanoic acid
PDGFR-βplatelet-derived growth factor receptor β
PFS progression-free survival
RAF rapidly accelerated fibrosarcoma
sEH soluble epoxide hydrolase
SIRT selective internal radiation treatment
SPE solid phase extraction
VEGFR vascular endothelial growth factor receptor
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