Chem 329

Metabolomics
Paper: Journal of Functional Foods

Uncovering the anti-metastasis effects

and mechanisms of capsaicin against
hepatocellular carcinoma cells by
metabolomics
 Background

Capsaicin, a kind of alkaloid from pepper cortex, has extensive

anti-tumor activities. However, the anti-metastasis effects and
mechanisms of capsaicin against hepatocellular carcinoma
(HCC) have not been deeply studied so far.

The anti-metastasis effect of capsaicin can be studied using a

metabolomics-based approach. Nontargeted
metabolomics approaches are being widely used for
carcinogenesis mechanism investigation and new drug
development.
Objectives

To study the anti-HCC effects of capsaicin

in human hepatoma SMMC-7721 and
HepG2 cells, and to explore its underlying
mechanisms based on untargeted
metabolomics approach.
Methodology

 Cell culture and treatment

 Cell viability assay
 Cell migration assay
 Cell invasion assay
 Cell adhesion assay
 1H NMR Analysis

 Data analysis
 Metabolic pathway analysis
 Multi-level network Analysis
 Statistical analysis
Methodology: Cell viability assay

 Employs MTT solution testing L02 cells, SMMC-7721

cells and HepG2 cells in a 96-well plates (5000–
6000) cells per well

 Different concentrations of capsaicin (0, 25, 50 and

100 μM) were used and tested at 24, 48 and 72 h
of incubation

 Absorbance was measured at 490 nm by using

Infinite M200 PRO plate reader (Tecan, Switzerland).
Methodology: Cell migration assay

 Used SMMC-7721 cells and HepG2 cells seeded in

6-well plates
 Artificial wound areas were created using pipette
tips
 PBS was used for washing and the cells were
treated with capsaicin (0, 25 and 50 μM) for 24 or
36 h
 Gaps were taken with a microscope and were
digitized using NIS-Elements software (Nikon, Ti-S,
Tokyo, Japan).
Methodology: Cell invasion assay

 Employs Transwell assay to evaluate the invasion

inhibitory effects of capsaicin in SMMC-7721 cells
and HepG2 cells

 600 μL of capsaicin (0, 25 and 50 μM) were

added to the lower compartments

 Sixteen fields were randomly selected to count the

number of invading cells in three independent
experiments
Methodology: Cell adhesion assay

 Capsaicin-treated cells were plated into gelatin-coated 24-

well plates and cultured at 37 °C for 0.5, 1, 1.5, 2 and 3 h

 After incubation, nonadherent cells were washed away by

warm PBS

 Adherent cells were fixed with 4% paraformaldehyde

solution, and stained with crystal violet.

 Intracellular stain was then solubilized with 1% SDS to

determine amount of adherent cells using the absorbance
value at 570 nm
Methodology: 1H NMR Analysis

 Used a Bruker 600-MHz AVANCE III NMR

spectrometer (Bruker, Germany)

 The 1H NMR spectra were acquired using water

suppression 1D noesygppr1d pulse sequence with
64 scans, 12345.7 Hz spectral width, 65,536
spectral size points, and a 1.0 s relaxation delay
Methodology: Data analysis

 MestReNova software was used for NMR Data analysis

 1H NMR chemical shifts in the spectra were referenced

to TSP

 Simca-P 13.0 software for principal component analysis

(PCA), partial least squares discriminant analysis (PLS-
DA) and orthogonal PLS-DA (OPLS-DA)

 The differential metabolites were identified according

to VIP-value (> 1) and t-test (p < 0.05).
Methodology: Metabolic pathway analysis

 Metabolic pathway was enriched and analyzed

using metaboAnalyst 3.0, a free web-based tool.

 The metabolites in the key pathways were imported

into Metscape to construct the metabolic network.
Methodology: Multi-level network construction

 The targets of capsaicin were gathered from Herbal Ingredients’

Targets (HIT) and ChemMapper Database

 Potential interactions between the candidate targets of capsaicin

and associated proteins were retrieved from String Database

 Differential metabolites were imported into Cytoscape Plug-in

MetScape to obtain the proteins associated with metabolites.

 Cytoscape software was used to construct a multi-level network,

consisted by candidate targets of capsaicin, interacting proteins
Methodology: Statistical analysis

 Graphs were generated using GraphPad Prism 5,

SIMCA-P 13.0, metaboAnalyst 3.0 and Cytoscape
3.7.0.

 Results were repeated at least three times, and the

values were expressed as mean ± SEM

 Differences were analyzed by one-way ANOVA for

multiple comparisons, and Student’s t test was used to
compare the control and capsaicin treatment groups
 Results and Discussion
Effects of capsaicin on the viability of human hepatoma cells

After 48 h or 72 h, it is shown that 25, 50 and 100 μM capsaicin

could dramatically inhibit the proliferation of both SMMC-7721
cells and HepG2 cells in a dose dependent
manner
 Results and Discussion
Effects of capsaicin on the viability of
human normal hepatic L02 cells
This was done to
test the potential
effects of
capsaicin on
normal
hepatocyte.
The results showed that 25, 50 and 100 μM capsaicin caused hardly
toxicity in L02 cells but larger doses of capsaicin (150 μM and 200
μM) suppressed the proliferation of L02 cells significantly and
might possess cytotoxicity to normal hepatocyte
 Results and Discussion
Effects of capsaicin on cell migration

Capsaicin significantly inhibited the migration of SMMC-7721

cells. Treatment with capsaicin (50 μM) for 36 h, decreased the
wound closure of SMMC- 7721 cells from 12.95% to 6.03%.
Results and Discussion
Effects of capsaicin on cell migration

Capsaicin also inhibited the migration of HepG2 cells.

Wound closure of HepG2 cells clearly decreased from
15.79% to 6.59%.
 Results and Discussion
Effects of capsaicin on invasion of human hepatoma cells

(a) (b)
Capsaicin (25 and 50 μM) dramatically inhibited the invasion of
SMMC-7721 cells (a) and HepG2 cells (b).
Results and Discussion
Effects of capsaicin on adhesion of human hepatoma cells
Cell-matrix adhesion is one of the important steps in cancer cell
metastasis and invasion

Adhesion was reduced dose-dependently after treatment with

capsaicin for 0.5, 1, 1.5, 2 and 3 h in SMMC-7721 cells.
 Results and Discussion
Effects of capsaicin on adhesion of human hepatoma cells

Adhesion was also reduced for HepG2 cells.

After treatment with capsaicin (50 μM) for 3 h, the adherence ratio of
SMMC- 7721 cells and HepG2 cells clearly reduced by 21.19% and
24.79%, respectively.
 Results and Discussion

Solid evidences are provided to establish that

capsaicin treatment:

 results in a decrease in cell migration and invasion

of human hepatoma cells
 significantly decreased the cell adhesion rate
 Results and Discussion
Multivariate statistical analysis
PCA can be applied as a pattern recognition method to discriminate
the different groups under unsupervised state.

The control and capsaicin treatment groups were separated clearly in

PCA scatter plot
 Results and Discussion
Multivariate statistical analysis

The PLS-DA model validation diagram indicated an excellent

predictive ability with all Q2 and R2-values lower than the original
points and the regression line of the Q2 -points intercepts a negative
value at the Y axis
Results and Discussion
Multivariate statistical analysis
OPLS-DA was used to identify and characterize metabolites order to
explore the changes of metabolites after capsaicin treatment
 Results and Discussion
Multivariate statistical analysis
The corresponding (V+S)-plot is a combination of VIP values and S-plot
plots and was utilized for seeking some endogenous metabolites
contributing to the separation
Results and Discussion
Multivariate statistical analysis
The levels of 14
metabolites were
significantly
changed in cells after
treatment with
capsaicin, including
higher levels of leucine,
valine, lactate, acetate
and lower levels of
threonine
Results and Discussion
Multivariate statistical analysis

4 metabolites were significantly changed in culture

supernatant after treatment with capsaicin, including
higher levels of succinate and pyroglutamate and lower
levels of lactate and ethanolamine
Results and Discussion
Pearson Correlation Coefficient Analysis
Explores the relationships between
the differential metabolites from
control and capsaicin treatment
groups
 The positive correlation between
threonine in cell and ethanolamine
in culture supernatant was strong
 The negative correlation with
other differential metabolites was
strong
 This indicates that the potential
biomarkers were interrelated
Results and Discussion
Metabolic pathways analysis

18 potential biomarkers
were used for pathway
analysis.

The results
of the MetPA pathway
analysis and metabolite
enrichment were consistent.
Results and Discussion
Metabolic pathways analysis

The most relevant metabolic

pathways regulated by
capsaicin included Glutathione
metabolism; Pyruvate
metabolism;
Phenylalanine metabolism;
Glycine, serine and threonine
metabolism;
Glycerophospholipid
metabolism.
 Results and Discussion
Metabolic pathways analysis

The metabolite-gene network was further established by Metscape to

better explain the bio-network of capsaicin for the anti-HCC effect.
 Results and Discussion

Metabolomics analysis was used to further

investigate the possible mechanisms of capsaicin
for the anti-metastasis effects.

Results revealed that capsaicin affected multiple

metabolic pathways, including glutathione
metabolism, pyruvate metabolism, phenylalanine
metabolism, and other metabolic pathways.
 Results and Discussion

Glutathione participates in glutathione metabolism

and is an important antioxidant in cells.

The glutathione was significantly elevated in

capsaicin treatment group compared to control
group, indicating that capsaicin helps protect
cells from oxidative stress.
 Results and Discussion

Capsaicin Treatment:
❖ may also disturb the degradation and metabolism of
fatty acids in cancer cells by affecting pyruvate
metabolism
❖ declined extracellular lactate content by affecting
lactate metabolism resulting to its anti-invasion and anti-
migration effects
❖ affects the energy metabolism pathway by affecting
succinate metabolism leading to an anti-metastasis
effect
 Results and Discussion

Capsaicin Treatment:
❖ cansignificantly increase the amount of intracellular
choline by affecting choline metabolism, indicating
that capsaicin can improve the lipid metabolism
disorder in HCC
 Results and Discussion
A multi-level network of capsaicin
to further explore the mechanisms of capsaicin

Capsaicin regulates glutathione metabolism, pyruvate metabolism,

phenylalanine metabolism, glycine, serine and threonine metabolism and
glycerophospholipid metabolism possibly through acting on 5 candidate targets
(VEGFA, CASP3, FOS, ABCB3, JUNB), thus affecting 19 interacting proteins, finally
resulting in the alteration of 9 metabolites and 5 metabolic pathways
 Results and Discussion

The multi-level network reveals that capsaicin

regulates glutathione possibly through acting on
GPx1, GPx4 and GPx7.

❖ reduced expression of GPx in HCC is positively correlated with

greater tumor size and higher recurrence rates
❖ capsaicin might inhibit the proliferation and invasion of HCC
cells by affecting the expression of GPx
❖ leading to an increase in glutathione and recovery from glutathione

metabolism disorders
Conclusion
 Capsaicin exhibited potential anti-metastasis effects through
remarkably suppressing cell proliferation, migration, and
invasion and hamperring cell-matrix adhesion in SMMC-7721
cells and HepG2 cells.

 1H NMR-based metabolomics results suggest 18 differential

metabolites were significantly changed inside and outside of
the cells after treatment of capsaicin, involved in glutathione
metabolism, pyruvate metabolism, phenylalanine metabolism,
etc

 multi-level network further uncovered the possible molecular

mechanisms of capsaicin in regulating these metabolites and
metabolic pathways