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\begin{document}

\title{Sevoflurane but not propofol enhances ovarian cancer cell malignancy
through regulating cellular metabolic and signalling mechanisms}



\author[1]{Cong Hu}%
\author[2]{Bincheng Wang}%
\author[2]{Zhigang Liu}%
\author[2]{Qiling Chen}%
\author[2]{Masashi Ishikawa}%
\author[1]{Han Lin}%
\author[1]{Qingquan Lian}%
\author[1]{jun li}%
\author[2]{Jia Li}%
\author[2]{Daqing Ma}%
\affil[1]{Wenzhou Medical University Second Affiliated Hospital}%
\affil[2]{Imperial College London}%


\vspace{-1em}



  
  \date{May 27, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background and Purpose: Surgery remains the first-line treatment of
ovarian cancer. However, perioperative risk factors including the choice
of anaesthetics may influence its recurrence after surgery. In the
current study, it was hypothesised that inhalational anaesthetic
sevoflurane and intravenous anaesthetic propofol might affect cancer
cellular metabolism and signalling, which might interfere the malignancy
of ovarian cancer cells. Experimental Approach: Cultured ovarian cancer
cells were exposed to 2.5\% sevoflurane or administered with 4 \selectlanguage{greek}μ\selectlanguage{english}g/mL
propofol for 2 hours followed by 24 hours recovery. Their cell
viability, proliferation, migration and invasion were assessed using
cell counting kit-8, Ki-67 staining, wound healing and Transwell assay.
Cellular signalling biomarkers were measured using immunofluorescent
staining and/or Western blot. Cultured media were collected for 1H-NMR
spectroscopy-based metabolomics analysis. Key Results: The cell
viability, proliferation, migration, and invasion of ovarian cancer
cells were enhanced by sevoflurane but suppressed by propofol.
Sevoflurane increased the GLUT1, MPC1, GLUD1, p-Erk1/2, and HIF-1\selectlanguage{greek}α
\selectlanguage{english}expressions but decreased the PEDF expression. In contrast to the
sevoflurane treatment, the ``mirror changes'' of these cellular markers
were observed with propofol. Sevoflurane increased levels of isopropanol
but decreased glucose and glutamine levels in the media, but the
opposite changes of those metabolites were found after propofol
treatment. Conclusion and Implications: These data indicated that unlike
propofol, sevoflurane enhanced ovarian cancer cell metabolism and
activated PEDF/Erk/HIF-1\selectlanguage{greek}α \selectlanguage{english}cellular signalling pathway, suggesting that
sevoflurane might have pro-tumour property but propofol might afford an
anti-tumour property. The translational value of this work warrants
further study.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Ovarian cancer is the sixth most frequently diagnosed cancer and the
seventh-highest mortality globally (Parkin, Bray, Ferlay, \& Pisani,
2005). There were an estimated more than 295,000 new cases and more than
184,000 deaths of ovarian cancer worldwide in 2018 (Bray et al., 2018).
The treatments for ovarian cancer include surgery, chemotherapy, and
radiotherapy. Among them, surgery remains the frontline therapy for
ovarian cancer patients, as the tumour resection at the early stage can
be very effective (Wyld, Audisio, \& Poston, 2015). However, ovarian
cancer recurrence following surgery is still a leading cause of death
(Saphner, Tormey, \& Gray, 1996; Zhu et al., 2011).

Many risk factors affect cancer recurrence, and one of them may be the
choice of anaesthetics and anaesthesia techniques (Horowitz, Neeman,
Sharon, \& Ben-Eliyahu, 2015). In an \emph{in vitro} study, oestrogen
receptor-positive or negative breast cancer cells were exposed to
sevoflurane, and the proliferation and migration of both types of cancer
cells were increased by sevoflurane (Ecimovic, McHugh, Murray, Doran, \&
Buggy, 2013). An previous publication from our laboratory demonstrated
that sevoflurane increased the migration of ovarian cancer
cells\emph{via} upregulating \emph{VEGFA, MMP11, CXCR2,} and
\selectlanguage{greek}\emph{TGF-β}\selectlanguage{english}gene expressions (Iwasaki et al., 2016). In contrary,
propofol was reported to have an ``anti-tumour'' property; for example,
our previous study demonstrated that propofol inhibited the
proliferation and migration of prostate cancer cells (Huang et al.,
2014). Clinically, a retrospective study reported that patients, who had
surgery for the breast, colonic or rectal cancer, were under
propofol-based total intravenous anaesthesia or sevoflurane inhalational
anaesthesia and their overall one-year and five-year survival rates were
higher with propofol anaesthesia (Enlund et al., 2014). However, the
underlying mechanisms are unknown.

Glucose is taken up by cancer cells through glucose transporter 1
(GLUT1) and transformed to pyruvate, which is converted to lactate
rather than enters mitochondria \emph{via} mitochondrial pyruvate
carrier 1 (MPC1) to be utilised by TCA cycle (Pezzuto, D'Ascanio, Ricci,
Pagliuca, \& Carico, 2020; Zou et al., 2019). Glutamine can be
transformed to be glutamate under the catalysation of glutamate
dehydrogenase 1 (GLUD1) for using in the TCA cycle which generates
crucial intermediates for cancer growth and survival (Craze et al.,
2019). Such process is named as glutaminolysis for which GLUD1 is the
key enzyme that locates in the inner membrane of mitochondria. Any
factors that affect the GLUD1 expression and activity may interfere
cancer cell survival (Son et al., 2013).

The transcriptional factor hypoxia-inducible factor-1 alpha (HIF-1\selectlanguage{greek}α\selectlanguage{english})
plays a key role of the development of many tumour types, including
breast, colonic and lung cancer (Zhao, Iwasaki, Yang, Savage, \& Ma,
2014) whilst the cellular signalling Erk1/2 pathway can regulate HIF-1\selectlanguage{greek}α
\selectlanguage{english}(Karagiota, Kourti, Simos, \& Mylonis, 2019). Several regulators can
regulate the signalling pathway of Erk1/2 and one of these regulators is
the pigment epithelium-derived factor (PEDF), a member of the serine
protease inhibitor family. The therapeutic values of PEDF were found for
choroidal neovascularisation, heart disease and cancer (Filleur, Nelius,
de Riese, \& Kennedy, 2009). Besides the upstream signalling of HIF-1\selectlanguage{greek}α,
\selectlanguage{english}the downstream effectors of HIF-1\selectlanguage{greek}α \selectlanguage{english}might also involve in the ovarian
cancer malignancy. C-X-C motif chemokine 12 (CXCL12), also called
stromal cell-derived factor 1 (SDF1), is originally correlated with the
activity of leukocytes and its receptor is C-X-C chemokine receptor type
4 (CXCR4), also named as fusin or cluster of differentiation 184 (CD184)
(Takano et al., 2014). It was found that CXCL12-CXCR4 was overexpressed
in a variety of cancer types, and is involved in the progression of
tumours (Scala et al., 2020).

In the current study, we hypothesised that unlike propofol, sevoflurane
might upregulate the expressions of GLUT1, MPC1, and GLUD1, which
increases the metabolism of ovarian cancer cells. The increased uptake
of glucose inhibits the expression of PEDF and, in turn, upregulates
p-Erk1/2, HIF-1\selectlanguage{greek}α, \selectlanguage{english}CXCL12, and CXCR4. The alterations of metabolism and
molecular entities by anaesthetics such as sevoflurane and propofol
might change the malignancy of ovarian cancer cells.

\textbf{Methods}

Cell culture

Ovarian cancer cell line (SKOV3) was purchased from ECACC (Wiltshire,
UK). SKOV3 cells were cultured in Gibco RPMI media 1640 (ThermoFisher,
Paisley, UK) supplemented with 10\% foetal bovine serum (FBS) and 1\%
penicillin-streptomycin (ThermoFisher, Paisley, UK) at 37\selectlanguage{ngerman}°C with 5\%
CO\textsubscript{2} and balanced with air. SKOV3 cells were administered
with 2.5\% sevoflurane (ABOTT, Sittingbourne, UK), 4 \selectlanguage{greek}μ\selectlanguage{english}g/mL propofol
(Sigma-Aldrich, Dorset, UK), media (na\selectlanguage{ngerman}ïve control), or intralipid
(vehicle control) (Santa Cruz Biotechnology, Dallas, Texas, USA) for 2
hours, and followed by 24 hours recovery in fresh media.

Cell viability was assessed with CCK-8 assay

After recovery, SKOV3 cells were incubated with media plus cell counting
kit-8 (CCK-8) solution at 37°C for 4 hours. The OD values of cancer
cells were detected at a wavelength of 450 nm. CCK-8 solution with
culture media but without cancer cells was used as blank. Cell viability
values equalled to {[}OD (treatment) - OD (blank){]} / {[}OD (control)
-- OD (blank){]} X 100\%.

Cell proliferation was assessed with Ki-67 staining

After recovery, SKOV3 cells were washed with phosphate-buffered saline
(PBS) (Santa Cruz Biotechnology, Dallas, Texas, USA) and incubated in
4\% paraformaldehyde (Santa Cruz Biotechnology, Dallas, Texas, USA) for
15 minutes. Cancer cells were washed with PBS plus 0.02\% Triton X-100
(Sigma-Aldrich, Dorset, UK) 3 times for 5 minutes each and blocked with
10\% donkey serum (Sigma-Aldrich, Dorset, UK) for 30 minutes. After
blocking, cells were incubated with anti-Ki-67 antibody (1:200, rabbit
polyclonal) (Santa Cruz Biotechnology, Dallas, Texas, USA) at 4°C
overnight and anti-rabbit antibody (1:1000, goat polyclonal) (Abcam,
Cambridge, UK) for 1 hour at room temperature under dark in sequence.
The 4',6-diamidino-2-phenylindole (DAPI) mounting media (Vector
Laboratories, Burlingame, California, USA) were mounted on cancer cells.
The fluorescent images were taken under fluorescence microscope and the
percentage of Ki-67 positive cells was calculated by Fiji (ImageJ 2.0)
software (National Institutes of Health, Bethesda, Maryland, USA).

Cell migration was assessed with wound healing assay

SKOV3 cells were seeded in petri dishes to form a continuous monolayer.
Cancer cells were incubated in pure media, 2.5\% sevoflurane, or 4 \selectlanguage{greek}μ\selectlanguage{english}g/mL
propofol for 2 hours. The monolayer of cancer cells was scratched to
form a cell-free gap. The petri dishes were washed with PBS to discard
suspended cancer cells. The cell-free gap was captured under a
microscope as the baseline. After incubated with culture media for 24
hours, the cell-free gap was captured again under the microscope to
calculate the migrate capability of cancer cells with Fiji (ImageJ 2.0)
software (National Institutes of Health, Bethesda, Maryland, USA).

Cell invasion was assessed with Transwell assay

SKOV3 cells were seeded in petri dishes and administered with media,
2.5\% sevoflurane, or 4 \selectlanguage{greek}μ\selectlanguage{english}g/mL propofol for 2 hours. After
administration, cancer cells were mixed with FBS-free media and seeded
in the upper chamber of the Transwell assay kit, which was pre-embedded
with Corning Matrigel matrix (Corning, New York, USA). The FBS-enriched
media was placed into the lower chamber of the Transwell assay kit.
After incubated at 37\selectlanguage{ngerman}°C for 24 hours, the upper chamber was inserted in
the 70\% methanol (ThermoFisher, Paisley, UK) for 30 minutes. The cancer
cells on the upper chamber were dyed with 0.1\% crystal violet
(Sigma-Aldrich, Dorset, UK) for 15 minutes and washed to get away the
leftover dye. The cells on the upper membrane of the upper chamber were
removed. The cells on the bottom membrane of it were regarded as
invasive cells and detected by a microscope. The number of invasive
cells was counted with Fiji (ImageJ 2.0) software (National Institutes
of Health, Bethesda, Maryland, USA).

GLUT1, MPC1, PEDF, HIF-1\selectlanguage{greek}α, \selectlanguage{english}CXCL12, and CXCR4 expressions were detected
with immunofluorescent staining

After recovery, SKOV3 cells were washed with phosphate-buffered saline
(PBS) (Santa Cruz Biotechnology, Dallas, Texas, USA) and incubated in
4\% paraformaldehyde (Santa Cruz Biotechnology, Dallas, Texas, USA) for
15 minutes. Cells were washed with PBS plus 0.02\% Triton X-100
(Sigma-Aldrich, Dorset, UK) 3 times for 5 minutes each and blocked with
10\% donkey serum (Sigma-Aldrich, Dorset, UK) for 30 minutes. After
blocking, cells were incubated with primary antibody at 4\selectlanguage{ngerman}°C overnight
and secondary antibody for 1 hour at room temperature under dark in
sequence (\textbf{Table S1} ). The 4',6-diamidino-2-phenylindole (DAPI)
mounting media (Vector Laboratories, Burlingame, California, USA) were
mounted on cancer cells. The expressions of proteins were detected by a
fluorescence microscope.

GLUT1, MPC1, GLUD1, PEDF, p-Erk1/2, Erk1/2, and HIF-1\selectlanguage{greek}α \selectlanguage{english}expressions were
determined with Western blot analysis

After recovery, SKOV3 cells were adapted with cell lysis buffer (Cell
signalling Technology, London, UK) to extract proteins. The protein
samples were mixed with NuPAGE LDS sample buffer (ThermoFisher, Paisley,
UK) and boiled at 95\selectlanguage{ngerman}°C for 10 minutes. The boiled samples were loaded
onto a NuPAGE Bis-Tris gel (ThermoFisher, Paisley, UK) for
electrophoresis. After electrophoresis, the protein on the gel was
transferred to a nitrocellulose membrane (ThermoFisher, Paisley, UK).
The membrane was blocked with 5\% non-fat milk for 1 hour and incubated
with primary antibody at 4°C overnight and secondary antibody on the
next day (\textbf{Table S1} ) for 1 hour and adapted with luminol
reagent solution A and B (Santa Cruz Biotechnology, Dallas, Texas, USA).
The protein bands were detected by GeneSnap Version 7.1 (Syngene,
Cambridge, UK) and the levels of protein were analysed by Fiji (ImageJ
2.0) software (National Institutes of Health, Bethesda, Maryland, USA).

The metabolic alterations in media were measured with proton NMR
spectroscopy

After recovery, the culture media of SKOV3 cells were collected. One day
before the NMR experiment, media samples were thawed at the room
temperature. The sample with 540 \selectlanguage{greek}μ\selectlanguage{english}L was collected and mixed with 60 \selectlanguage{greek}μ\selectlanguage{english}L
potassium phosphate buffer (pH = 7.4) containing 1.5 M
KH\textsubscript{2}PO\textsubscript{4}, 1mM NaN\textsubscript{3}, 0.1\%
TSP and D\textsubscript{2}O. A total of 580 \selectlanguage{greek}μ\selectlanguage{english}L mixture was placed into
an NMR tube (Bruker Corporation, Rheinstetten, Germany) with an outer
diameter of 5 mm. \textsuperscript{1}H-NMR spectra of media samples were
obtained using a Bruker 600 MHz spectrometer (Bruker Corporation,
Rheinstetten, Germany) at the operating \textsuperscript{1}H frequency
of 600.13 MHz at a temperature of 300 K. A standard NMR pulse sequence
(recycle delay-90\selectlanguage{ngerman}º-t\textsubscript{1}-90º-t\textsubscript{m}-90º
acquisition) was applied to acquire \textsuperscript{1}H-NMR spectral
data (t\textsubscript{1} = 3 \selectlanguage{greek}μ\selectlanguage{english}s, t\textsubscript{m} = 100 ms). The water
peak suppression was achieved using selective irradiation during a
recycle delay of 4 s and t\textsubscript{m}. A 90\selectlanguage{ngerman}º pulse was adjusted to
\textasciitilde{}10 \selectlanguage{greek}μ\selectlanguage{english}s. A total of 32 scans for cell media were
collected into 64 k data points with a spectral width of 20
ppm.\textsuperscript{1}H-NMR spectral data were acquired using TopSpin
4.0.9 (Bruker Corporation, Rheinstetten, Germany). The spectral data
were imported into MATLAB R2018a (MathWorks, Natick, Massachusetts, USA)
and SIMCA (Sartorius, Gottingen, Germany) for multivariate statistical
analysis. The chemical shift ranged from 4.7 to 5.0, and from -1 to 0.3
was cut. The spectral data was aligned with recursive segment-wise peak
alignment method and normalised with probabilistic quotient
normalisation method (Dieterle, Ross, Schlotterbeck, \& Senn, 2006;
Veselkov et al., 2009). Metabolites were identified using Chenomx
software (CHENOMX, Edmonton, Canada).

Materials

The SKOV3 cell line was purchased from ECACC, Wiltshire, UK. The Gibco
RPMI media 1640, penicillin-streptomycin, methanol, NuPAGE LDS sample
buffer, NuPAGE Bis-Tris gel and nitrocellulose membrane were obrained
from ThermoFisher, Paisley, UK. The sevoflurane was purchased from
ABOTT, Sittingbourne, UK. The propofol, Triton X-100, donkey serum and
crystal violet were acquired from Sigma-Aldrich, Dorset, UK. The
intralipid, PBS, paraformaldehyde, anti-Ki-67 antibody and luminol
reagent solution were from Santa Cruz Biotechnology, Dallas, Texas, USA.
The anti-rabbit and anti-mouse antibodies for immunofluorescent
staining, anti-GLUT1, anti-GLUD1, anti-PEDF, anti-CXCL12 and anti-CXCR4
antibodies were obtained from Abcam, Cambridge, UK. The DAPI mounting
media was acquired from Vector Laboratories, Burlingame, California,
USA. The Corning Matrigel matrix was purchased from Corning, New York,
USA. The anti-MPC1 and anti-HIF-1\selectlanguage{greek}α \selectlanguage{english}antibodies were obtained from Novus
Biologicals, Abingdon, UK. The anti-p-Erk1/2 and anti-Erk1/2 antibodies,
anti-rabbit and anti-mouse antibodies for Western blot analysis, and
cell lysis buffer were acquired from Cell signalling Technology, London,
UK. The anti-GAPDH antibody was purchased from Merck, Hertfordshire, UK.
The NMR tube was from Bruker Corporation, Rheinstetten, Germany.

\textbf{Results}

Effects of sevoflurane or propofol on cancer malignancy in ovarian
cancer cells

Sevoflurane significantly increased the ovarian cancer cell viability of
compared to na\selectlanguage{ngerman}ïve control (NC \emph{vs} . S, 100.0 ± 1.5 \emph{vs} .
122.0 ± 4.1, p \textless{} 0.0001, n = 6), while propofol significantly
decreased the cell viability (NC \emph{vs} . S, 100.0 ± 1.5 \emph{vs} .
61.6 ± 5.4, p \textless{} 0.0001, n = 6) (\textbf{Fig 1A} ). The
proliferation of SKOV3 cells was indicated by the number of Ki-67
positive cells (\textbf{Fig 1B} ). It was found that the number of Ki-67
positive cells was significantly increased by sevoflurane administration
(NC \emph{vs} . S, 32.0 ± 5.1 \emph{vs} . 52.4 ± 13.8, p \textless{}
0.01, n = 6). However, Ki-67 positive cells were significantly decreased
by propofol treatment (NC \emph{vs} . P, 32.0 ± 5.1 \emph{vs} . 16.0 ±
7.3, p \textless{} 0.05, n = 6) (\textbf{Fig 1C} ).

The migrate capability of ovarian cancer cells after sevoflurane
treatment was significantly greater than that of the naïve control
(NC\emph{vs} . S, 55.2 ± 7.5 \emph{vs} . 92.5 ± 8.2, p \textless{}
0.0001, n = 6) (\textbf{Fig 1D} ). In contrast, the migrate capability
of cancer cells was inhibited by propofol exposure (NC \emph{vs} . P,
55.2 ± 7.5\emph{vs} . 34.3 ± 9.4, p \textless{} 0.01, n = 6)
(\textbf{Fig 1E} ). The invasion of ovarian cancer cells was evaluated
by comparing the number of invasive cells with Transwell assay.
Significantly higher number of invasive cells was observed in the
sevoflurane group compared to naïve control (NC \emph{vs} . S, 1.0 ± 0.1
\emph{vs} . 2.2 ± 0.6, p \textless{} 0.0001, n = 6) (\textbf{Fig 1F} ),
whereas propofol exhibited an opposite effect (NC \emph{vs} . P, 1.0 ±
0.1 \emph{vs} . 0.4 ± 0.1, p \textless{} 0.05, n = 6) (\textbf{Fig 1G}
).

Effects of sevoflurane or propofol on the expressions of GLUT1, MPC1,
and GLUD1 in ovarian cancer cells

Based on the immunofluorescent staining of cells, the intensities of
GLUT1 (\textbf{Fig 2A} ) and MPC1 (\textbf{Fig 2B} ) were higher in the
sevoflurane group compared to naïve control, but much lower in the
propofol group than the naïve control. From Western blot analysis, the
protein expression levels of GLUT1 (NC \emph{vs} . S, 1.0 ± 0.1\emph{vs}
. 1.4 ± 0.3, p \textless{} 0.01, n = 6) and MPC1 (NC \emph{vs} . S, 1.0
± 0.3 \emph{vs} . 1.5 ± 0.2, p \textless{} 0.01, n = 6) were
significantly increased after sevoflurane exposure \textbf{(Fig 2C} ).
However, the expression levels of GLUT1 (NC \emph{vs} . P, 1.0 ±
0.1\emph{vs} . 0.3 ± 0.1, p \textless{} 0.0001, n = 6) and MPC1
(NC\emph{vs} . P, 1.0 ± 0.3 \emph{vs} . 0.6 ± 0.3, p \textless{} 0.05, n
= 6) were significantly decreased after propofol treatment compared to
the naïve control (\textbf{Fig 2E and F} ). Similarly, the expression
level of glutamate dehydrogenase 1 (GLUD1) was significantly increased
by sevoflurane administration (NC \emph{vs} . S, 1.0 ± 0.1 \emph{vs} .
1.4 ± 0.1, p \textless{} 0.01, n = 6), but decreased by propofol
administration (NC \emph{vs} . P, 1.0 ± 0.1 \emph{vs} . 0.6 ± 0.2, p
\textless{} 0.001, n = 6) (\textbf{Fig 2D} ).

Effects of sevoflurane or propofol on the cellular metabolism of SKOV3
cells

The metabolic profiles obtained from media samples of naïve control,
vehicle control, sevoflurane, and propofol groups were analysed using
unsupervised PCA analysis with 2 principal components. The PCA scores
plot showed a clear grouping pattern among these four groups
(\textbf{Fig 3A} ). Pair-wise comparisons of naïve control \emph{vs} .
sevoflurane group and vehicle control \emph{vs} . propofol group were
carried out using OPLS-DA analysis with one predictive component and one
orthogonal component. Permutation p values showed both models of naïve
control \emph{vs} . sevoflurane (p = 0.005) and vehicle control\emph{vs}
. propofol (p = 0.005) had significances. The permutation p values
together with R\textsuperscript{2}X, Q\textsuperscript{2}X, and
Q\textsuperscript{2}Y of two models were summarised (\textbf{Table 1} ).
The OPLS-DA scores plots showed there were clear separations between
naïve control and sevoflurane group (\textbf{Fig 3B} ), and vehicle
control and propofol group (\textbf{Fig 3D} ). The OPLS-DA loadings
plots showed that the concentrations of glucose and glutamine were
decreased, and the concentration of isopropanol was increased in the
sevoflurane group compared to the naïve control group (\textbf{Fig 3C}
). However, the concentrations of glucose and glutamine were increased,
and the concentration of isopropanol was decreased in the propofol group
in contrast to vehicle control group (\textbf{Fig 3E} ). After
anaesthetics administration, the concentrations of lactate, pyruvate,
acetate, alanine, valine, and leucine were increased by both sevoflurane
and propofol. Furthermore, propofol also increased the levels of
glycerol, fatty acids, asparagine, succinate, acetone, arginine, and
isoleucine, but decreased the level of ethanol (\textbf{Table 2} ).

Regulations of sevoflurane or propofol on PEDF expression, Erk pathway,
and HIF-1\selectlanguage{greek}α \selectlanguage{english}expression

From immunofluorescent staining, it was found that the fluorescent
intensity of pigment epithelium-derived factor (PEDF) was decreased in
the sevoflurane group but increased in the propofol group compared to
na\selectlanguage{ngerman}ïve control (\textbf{Fig 4A} ). The result was validated using Western
blot analysis, which showed the significantly lower expression level of
PEDF after sevoflurane exposure (NC \emph{vs} . S, 1.0 ± 0.1 \emph{vs} .
0.6 ± 0.2, p \textless{} 0.0001, n = 6) but significantly higher after
propofol exposure (NC \emph{vs} . P, 1.0 ± 0.1 \emph{vs} . 1.2 ± 0.1, p
\textless{} 0.05, n = 6) (\textbf{Fig 4D} ). Different from PEDF
expression levels, the Western blot analysis showed that the expression
level ratio of p-Erk1/2 to Erk1/2 was significantly increased in the
sevoflurane group (NC \emph{vs} . S, 1.0 ± 0.1 \emph{vs} . 1.3 ± 0.2, p
\textless{} 0.05, n = 6), but decreased in the propofol group compared
to the control (NC \emph{vs} . P, 1.0 ± 0.1 \emph{vs} . 0.7 ± 0.2, p
\textless{} 0.05, n = 6) (\textbf{Fig 4E} ). Similarly, the fluorescent
staining showed SKOV3 cells administered with sevoflurane had a higher
intensity of hypoxia-inducible factor 1-alpha (HIF-1\selectlanguage{greek}α\selectlanguage{english}) than the na\selectlanguage{ngerman}ïve
control, while those of the propofol group had a lower intensity of
HIF-1\selectlanguage{greek}α \selectlanguage{english}than control (\textbf{Fig 4B} ). From Western blot analysis, it
was also found that the expression level of HIF-1\selectlanguage{greek}α \selectlanguage{english}was significantly
increased after cancer cells administered with sevoflurane (NC\emph{vs}
. S, 1.0 \selectlanguage{ngerman}± 0.3 \emph{vs} . 1.5 ± 0.5, p \textless{} 0.05, n = 6), but
significantly decreased after propofol treatment compared to the control
(NC vs. S, 1.0 ± 0.3 vs. 0.4 ± 0.3, p \textless{} 0.05, n = 6)
(\textbf{Fig 4F} ).

Effects of sevoflurane or propofol on CXCL12 and CXCR4 expressions

According to immunofluorescent staining, the intensity of C-X-C motif
chemokine ligand 12 (CXCL12) was increased in the sevoflurane group but
decreased in the propofol group (\textbf{Fig 5A} ). In similar, the
fluorescent staining of C-X-C motif chemokine receptor 4 (CXCR4) was
higher in the sevoflurane group than the control, and the intensity of
the marker was lower in the propofol group than the control (\textbf{Fig
5B} ). These results showed the expressions of CXCL12 and CXCR4 were
both increased by sevoflurane but decreased by propofol administration.

\textbf{Discussion}

Our current study suggested that inhalational anaesthetic sevoflurane
enhanced ovarian cancer cell viability, proliferation, migration, and
invasion. In contrast, intravenous anaesthetic propofol inhibited those
cellular activities. These phenotypic observations could be associated
with upregulated expressions of GLUT1, MPC1, GLUD1, p-Erk1/2, HIF-1\selectlanguage{greek}α,
\selectlanguage{english}CXCL12 and CXCR4, and downregulated PEDF expression in the sevoflurane
group. Sevoflurane, but not propofol, promoted metabolism of ovarian
cancer cells by enhancing the uptake of the metabolic substrates such as
glucose and glutamine.

The lower concentration of glucose and higher concentration of pyruvate
in the media were found after sevoflurane exposure compared to the na\selectlanguage{ngerman}ïve
control, which were in agreement with observed upregulated expression
level of GLUT1. The enhanced activity of GLUT1 may transport more
glucose into the cytoplasm to convert to pyruvate, which explained the
metabolic changes in the media after sevoflurane administration. In
contrast, propofol downregulated GLUT1, which resulted in the less
uptake of glucose from media, and hence the concentration of glucose was
increased after propofol exposure. The cellular glucose uptake\emph{via}
high level of GLUT1 expression was correlated with the malignancy of
cancers (Leung, 2004; Pezzuto et al., 2020) and the overexpression of
GLUT1 in cancer cells were essential for the high rate of glycolysis
(Wright, 2020). Propofol was reported to downregulate\emph{GLUT1} gene
in macrophages (Tanaka et al., 2010) and this was in line with the
downregulation of GLUT1 proteins in the rat brain tissue under hypoxic
preconditioning (Xiao et al., 2020). This is quite similar to cancer
cell scenario, as, under high proliferative rate, cancer cells had
inadequate oxygen supply and were actually under hypoxic condition
(Mudassar, Shen, O'Neill, \& Hau, 2020). These supported our findings
that the malignancy of ovarian cancer cells was related to the
expression of GLUT1 after anaesthetic administration.

The concentration of lactate was increased in both sevoflurane and
propofol treatments, which might be resulted from the increased pyruvate
in glycolysis and then generated more lactate. It was reported that
sevoflurane and propofol both increased the lactate level in the blood
of dogs (Söbbeler et al., 2018). Another study in mice also demonstrated
that sevoflurane increased pyruvate and lactate levels (Horn \& Klein,
2010). All these reports were in line with the findings of our current
study.

It was found that both the expressions of MPC1 and GLUD1 were
upregulated after sevoflurane exposure but were downregulated after
propofol treatment. Besides, the concentration of glutamine in media was
decreased after sevoflurane administration. MPC1, a member of
mitochondrial carrier system, locates at the inner membrane of
mitochondria and transports pyruvate into mitochondria from cytoplasm
(Taylor, 2017). The expression of MPC1 is decreased in most tumour types
especially those under a high rate of proliferation as related to the
increased rate of glycolysis. This suggests that pyruvate likely shifts
from the mitochondrial TCA cycle to cytoplasm glycolysis, which does not
require oxygen supply as cancer cells are usually under hypoxic
condition (Rauckhorst \& Taylor, 2016). In addition, glutaminolysis
compensates for the disturbed function of the TCA cycle due to less
pyruvate intake and cancer cells, in turn, uptake glutamine and convert
them into glutamate under the activation of GLUD1. The glutamate can be
used by TCA cycle to restore the survival of cancer cells as the
intermediates of TCA cycle are the source for synthesis of amino acids,
proteins, fatty acids, lipids, carbon skeleton, and nucleic acids (Yoo,
Yu, Sung, \& Han, 2020). Evidence from other studies showed propofol
might disturb the mitochondrial respiratory chain, which was related to
TCA cycle (Berndt et al., 2018). It was also reported that unlike
inhibitory effects of propofol, sevoflurane preserved the function of
the mitochondrial respiratory chain in a myocardial ischaemic model
(Lotz, Stumpner, \& Smul, 2020). Our data demonstrated that after
sevoflurane administration, the MPC1 and GLUD1 expressions were
upregulated, which might enhance the activity of the TCA cycle to meet
the demands of cancer survival and progression. The decreased
concentration of glutamine in media suggested that the utilisation of
glutamine was likely increased and glutaminolysis was then promoted.
With the disturbed function of the TCA cycle after propofol exposure,
amino acids that can be used in the TCA cycle were accumulated, such as
asparagine and arginine (Pasini et al., 2018), which was consistent with
our findings.

Except glucose and glutamine, another ``mirror change'' of metabolites
between sevoflurane and propofol administration was isopropanol. It was
reported that the level of isopropanol was increased in the exhaled
breath of lung cancer patients, and it had been regarded as a potential
biomarker for lung cancer diagnosis (Chien et al., 2017). It seemed the
level of isopropanol had some correlations with cancer malignancy, which
was consistent with the findings of this study that sevoflurane enhanced
the malignancy of ovarian cancer cells and increased the level of
isopropanol, while propofol inhibited the malignancy of ovarian cancer
cells and decreased the level of isopropanol. Isopropanol can be
reversibly converted to acetone (Beauchamp, Valento, \& Kim, 2016; Li,
Liu, Liu, Cheng, \& Duan, 2017), which may also contribute to the
increased level of acetone in the media of the propofol group.

The levels of glycerol and fatty acids were increased in the propofol
group. Through \selectlanguage{greek}β-\selectlanguage{english}oxidation of fatty acids, acetyl-CoA is generated and
used in the TCA cycle (Y. Liu, 2006). Thus, the changes of glycerol and
fatty acids in the propofol group were another evidence that the
mitochondria function and TCA cycle was inhibited or disturbed by
propofol treatment. The glycerol and fatty acids might also come from
cell membrane degradation and phospholipids broke down into them. From
an earlier study, it was found that propofol affected the membrane
ultrastructure of HeLa cells that the surface roughness of cellular
membrane was decreased in a dose-dependent manner (Zhang et al., 2016).

In the current study, the expression level of PEDF was decreased after
sevoflurane administration but increased after propofol treatment. In
human retinal pigment epithelium, the expression of GLUT1 was increased
under hypoxia condition that resulted in the increased uptake of
glucose, which led to a decrease of PEDF expression (Calado, Alves,
Sim\selectlanguage{ngerman}ão, \& Silva, 2016). Another study also reported that the
overexpression of the \emph{PEDF} gene in mice was related to the
reduction of glucose uptake and decreased expression of GLUT1 (Calado,
Diaz-Corrales, \& Silva, 2016). These reports were consistent with our
findings that sevoflurane increased the GLUT1 expression and glucose
uptake, which led to a downregulated expression of PEDF. However, an
opposite effect was found with propofol treatment.

In the current study, Erk1/2 signalling pathway was induced after
sevoflurane exposure but inhibited after propofol exposure. There was
evidence that the increased expression of PEDF was related to the
inhibition of Erk1/2 signalling pathway in a diabetic model (Dong et
al., 2019), which was in line with our results. HIF-1\selectlanguage{greek}α \selectlanguage{english}is a
transcriptional factor that can be regulated by a variety of signalling
pathways, and Erk1/2 pathway is one of them (R. M. Liu, Xu, Chen, Feng,
\& Xie, 2020). In cancer cells, the HIF-1\selectlanguage{greek}α \selectlanguage{english}is overexpressed, which
regulates tumour survival-related genes, such as \emph{CXCL12}
and\emph{CXCR4} (Gola et al., 2020; Xue et al., 2020). It was in line
with the results of the current study that sevoflurane upregulated
Erk1/2 signalling pathway, and HIF-1\selectlanguage{greek}α, \selectlanguage{english}CXCL12 and CXCR4 expressions,
while propofol downregulated these molecular entities.

Our study has some limitations. Firstly, the causal relationship between
cellular signalling changes and metabolic alterations induced by
anaesthetics remains unknown. However, it is very likely that, for
example, sevoflurane promotes cancer cell survival and development due
to survival cellular signalling pathway activation whereby more energy
substrates use up for cell proliferation and growth. Secondly, our
cultured cell study may not be relevant to human. Therefore, the
implications of our current study may be limited. However, in some
clinical studies, breast, colonic and rectal cancer patients were
anaesthetised with inhalational anaesthetics sevoflurane or desflurane,
or intravenous anaesthetic propofol during surgery and the survival rate
of propofol anaesthetised patients were significantly higher than those
with inhalational anaesthesia (Enlund et al., 2014; Wu et al., 2018).
Laboratory data including the one reported here and retrospective
clinical data all point to that sevoflurane might be a risk factor for
cancer patients, while propofol may be beneficial to cancer patients for
their surgery. Therefore, clinical studies are urgently needed to
evaluate anaesthesia regimens for cancer patients to optimise the
surgical outcomes.

Conclusions

The current study demonstrated that sevoflurane upregulated GLUT1, MPC1,
and GLUD1 expressions of ovarian cancer cells, while propofol
downregulated the expressions of these molecules. These regulations by
sevoflurane or propofol on ovarian cancer cells led to different
metabolic features. The upregulated GLUT1 expression and, in turn,
increased glucose uptake after sevoflurane exposure resulted in a
decreased expression of PEDF. In opposite, an increased expression of
PEDF was identified after propofol treatment. Furthermore, in contrast
to propofol, sevoflurane upregulated Erk1/2 pathway, HIF-1\selectlanguage{greek}α, \selectlanguage{english}CXCL12 and
CXCR4 expressions through PEDF inhibition \emph{per se} . In summary,
the profiling alterations of molecular and metabolic modulations found
in the present study indicate the pro- and anti-tumour properties of
sevoflurane and propofol, respectively. The translational value of these
is subjected to study further in clinical settings.

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\textbf{Figure Legends}

\textbf{Fig 1. The cell viability, proliferation, migration, and
invasion of ovarian cancer cells after sevoflurane or propofol
administration.}

Ovarian cancer (SKOV3) cells were administered with pure culture media
(na\selectlanguage{ngerman}ïve control), 2.5\% sevoflurane, or 4 \selectlanguage{greek}μ\selectlanguage{english}g/mL propofol. The cell
viability of ovarian cancer cells was evaluated with cell counting kit-8
assay (A). The cell proliferation of ovarian cancer cells was evaluated
by identifying Ki-67 positive cells (B and C). The migration of SKOV3
cells was evaluated by calculating gap closure with wound healing assay
(D and E). The invasion was evaluated by identifying invasive cells with
Transwell assay (F and G). The data was expressed as mean \selectlanguage{ngerman}± standard
deviation and dots plot (n = 6). *p \textless{} 0.05, **p \textless{}
0.01, ****p \textless{} 0.0001 versus naïve control. Scale bar: 100 \selectlanguage{greek}μ\selectlanguage{english}m.
NC, na\selectlanguage{ngerman}ïve control group; S, sevoflurane group; P, propofol group.

\textbf{Fig 2. The effects of sevoflurane and propofol on the
expressions of GLUT1, MPC1, and GLUD1 in ovarian cancer cells.}

SKOV3 cells were exposed to media (naïve control), 2.5\% sevoflurane, or
4 \selectlanguage{greek}μ\selectlanguage{english}g/mL propofol. The expressions of GLUT1 (A) and MPC1 (B) were
detected with immunofluorescent staining. The expression levels of these
two markers were validated with Western blot analysis and the expression
level of GLUD1 was also analysed (C). The intensity of the bands in
Western blot analysis was normalised by housekeeping protein GAPDH. The
data was analysed with one-way ANOVA and Dunnett multi-comparison test
(D-F) and was presented as mean \selectlanguage{ngerman}± standard deviation and dots plot (n =
6). *p \textless{} 0.05, **p \textless{} 0.01, ***p \textless{} 0.001,
****p \textless{} 0.0001 versus naïve control. Scale bar: 50 \selectlanguage{greek}μ\selectlanguage{english}m. NC:
na\selectlanguage{ngerman}ïve control group; S: sevoflurane group; P: propofol group; GLUT1:
glucose transporter 1; MPC1: mitochondrial pyruvate carrier 1; GLUD1:
glutamate dehydrogenase 1; ANOVA: analysis of variance.

\textbf{Fig 3. The metabolomics study of sevoflurane- or
propofol-treated ovarian cancer cells using \textsuperscript{1}H-NMR
spectroscopy.}

Ovarian cancer cells were administered with media (naïve control),
intralipid (vehicle control), 2.5\% sevoflurane or 4 \selectlanguage{greek}μ\selectlanguage{english}g/mL propofol for
2 hours plus 24 hours recovery time. After treatment, the culture media
of ovarian cancer SKOV3 cells was collected for \textsuperscript{1}H-NMR
spectroscopy experiment. PCA scores plot of \textsuperscript{1}H-NMR
spectra of media was shown (A). R\textsuperscript{2}X represented the
fraction of variance in the \textsuperscript{1}H-NMR data modelled by
two principal components (t{[}1{]} and t{[}2{]}). The OPLS-DA scores
plots (B: NC \emph{vs} . S; D: VC \emph{vs} . P) and loadings plots (C:
NC\emph{vs} . S; E: VC \emph{vs} . P) were derived
from\textsuperscript{1}H-NMR spectral data (n = 10). The colour bar
indicated the correlation coefficient values (r\textsuperscript{2}) to
be high in red and low in blue. NC: na\selectlanguage{ngerman}ïve control group; VC: vehicle
control group; S: sevoflurane group; P: propofol group; PCA: principal
component analysis; OPLS-DA: orthogonal projection to latent
structures-discriminant analysis; Glc: glucose; Lac: lactate; Pyr:
pyruvate; Gln: glutamine; Ace: acetate; Ala: alanine; IPA: isopropanol;
Val: valine; Leu: leucine; Eth: ethanol; Gro: glycerol; Asn: asparagine;
FA: fatty acids; Suc: succinate; Acn: acetone; Arg: arginine; Ile:
isoleucine.

\selectlanguage{greek}\textbf{Fig 4. The opposite effects of sevoflurane and propofol on
PEDF/Erk/HIF-1α pathway in ovarian cancer cells.}\selectlanguage{english}

Ovarian cancer cells were administered with media (na\selectlanguage{ngerman}ïve control), 2.5\%
sevoflurane, or 4 \selectlanguage{greek}μ\selectlanguage{english}g/mL propofol. The expressions of PEDF (A) and HIF-1\selectlanguage{greek}α
\selectlanguage{english}(\selectlanguage{greek}Β\selectlanguage{english}) were detected with immunofluorescence. The expression levels of
PEDF, p-Erk1/2, Erk1/2, and HIF-1\selectlanguage{greek}α \selectlanguage{english}were evaluated with Western blot
analysis (C). The intensity of PEDF and HIF-1\selectlanguage{greek}α \selectlanguage{english}bands was normalised by
GAPDH (D and F). While the intensity of p-Erk1/2 band was normalised by
Erk1/2 (E). Data was analysed with one-way ANOVA with Dunnett
multi-comparison test and presented as mean \selectlanguage{ngerman}± standard deviation and
dots plot (n = 6). *p \textless{} 0.05, ****p \textless{} 0.0001 versus
naïve control. Scale bar: 50 \selectlanguage{greek}μ\selectlanguage{english}m. NC: na\selectlanguage{ngerman}ïve control group; S: sevoflurane
group; P: propofol group; PEDF: pigment epithelium-derived factor;
p-Erk1/2: phospho-extracellular-signal-regulated kinase 1 and 2; HIF-1\selectlanguage{greek}α:
\selectlanguage{english}hypoxia-inducible factor-1 alpha; ANOVA: analysis of variance.

\textbf{Fig 5. The expressions of CXCL12 and CXCR4 in ovarian cancer
cells after sevoflurane or propofol exposure.}

SKOV3 cells were exposed to pure culture media (na\selectlanguage{ngerman}ïve control), 2.5\%
sevoflurane, or 4 \selectlanguage{greek}μ\selectlanguage{english}g/mL propofol for 2 hour and followed by 24 hours
recovery. The expressions of CXCL12 (A) and CXCR4 (B) were detected with
immunofluorescent staining (green) and overlaid with DAPI (blue).
Sevoflurane exposure increased the expressions of CXCL12 and CXCR4 in
ovarian cancer cells, while propofol decreased the expressions of both
markers. NC: na\selectlanguage{ngerman}ïve control group; S: sevoflurane group; P: propofol
group; CXCL12: C-X-C motif chemokine ligand 12; CXCR4: C-X-C motif
chemokine receptor 4.\selectlanguage{english}
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