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APOPTOSIS IN CANCER CELL LINES USING METABOLOMICS

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Guillem Climent

on 31 October 2013

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Transcript of APOPTOSIS IN CANCER CELL LINES USING METABOLOMICS

APOPTOSIS IN CANCER CELL LINES USING METABOLOMICS
Discussion
Introduction
Results
Materials
and
methods

Vicente Barberá Navarro
Gracián Camps Ramón
Guillermo Climent Gargallo
Anda Gabriela Marsavela
Biotechnology Degree
4th Year
Group 4I3

Politechnic University of Valencia
Cell Culture:
- Human embryonic kidney (
HEK293
)
- Human hepatocellular carcinoma (
HepG2
)




Culture Conditions:
- Humidified atmosphere
- 5% CO2
- 37 Celsius
- DMEM supplemented with 10% FBS
DETECTION OF VIABILITY:
MTT assay
- Absorbance-based assay:

MTT
(yellow tetrazole)
Formazan
(purple)
Stimulation of apoptosis:
- Replacement of growth medium with a fresh one containing
5-FU
or
etoposide
in 1% DMSO

Stimulation of necrosis:
-
Heating
of the cells for 20 minutes at 57 Celsius on a hot plate, after replacement of medium with fresh growth medium containing 1% DMSO

Control cells:
- Fresh growth medium containing 1% DMSO
without any further treatment

Treated cell were subsequently incubated for
18
,
24
,
48
and
72
hours before analysis
DETECTION OF APOPTOSIS:
Caspase 3/7 assay
- [
Caspase-3
] and [
Caspase-7
]
- Represents % of apoptosis in cell

Sample preparation for the NBS assay:
- Cuantification of 42 metabolites in blood sample was adapted for this study
Metabolite detection and quantification

- Derivatized amino acids by
neutral loss scanning
- Data evaluation performed using

ChemoView
software (AB Sciex)
Statistical Analysis
Metabolomic techniques allowed:
The study of many types of cancer...

... and their
specific metabolites
and biomarkers
Prostate
,
breast
,
ovary
,
colorectum
,
colon
,
kidney
,
lung
,
bladder
,
leukaemia
...
What is measured?
Cancer metabolism is studied in detail to monitor the targeted treatment of the disease.

The Paper
This paper describes a
metabolomics approach
to investigate changes in the small molecule composition of apoptotic and necrotic cells
Aim:
to reveal and determine
new

apoptosis biomarkers
in cell culture models
MATERIALS:
-
Apoptosis inducers
(
5-FU
and
etoposide
)

TECHNIQUES:
- Newborn screening assay (
NBS
)
- Flow injection analysis (
FIA
)
- Neutral loss scan (
tandem MS
): API4000 mass spectrometer
Statistical analysis was performed using the public
metaP
server at the Helmholtz Center Munich, which provides
automated and standarized data analysis for quantitative metabolomic data

Association between
metabolite concentrations and multiclass categorical phenotypes
was tested using:

- Principal Component Analysis (
PCA
)
-
Kruskal-Wallis tests
-
P-value of <0.05
after Bonferroni correction
Implementation of metabolomics in apoptosis studies in order to detect several biomarkers.

Problems:
-
Complexity of the metabolic profile
of a whole organism
- Molecule composition is strongly affected by
several factors
- Low sensitivity due to the use of
NMR
to metabolite determination

Solutions:
- Use a simple and robust
LC/MS
based system suitable for high-throughput analysis the newborn screening (
NBS
) assay (
higher sensitivity
)
- Studies in
cell culture
are easier to control than those in whole organisms in regard to complexity application of metabolomics
DISCUSSION
Solution:
To be able to find metabolites affected specifically by the
apoptotic process
, we extended our experimental set-up to further
pro-apoptotic agents
:
etoposide
and
5-fluorouracil
(5-Fu) and compared the apoptosis-induced metabolic alterations with previous results
New problem:
the observed metabolic changes could be a
consequence of apoptosis
,
other staurosporine specific affects
or
the combination of both
Amongst the 42 measured metabolites, only
alanine and glutamate

were found to be significantly regulated
regardless of whether apoptosis was induced by staurosporine
,
etoposide
or
5-Fu
We propose that the increase in concentrations of alanine and glutamate in apoptotic cells may be associated with
taurine metabolism
- Our results demonstrate that
the NBS assay
, after adaptation and optimization,
is suitable for metabolomic studies at the cellular level

-
The number (42) of measured metabolites is sufficient
to determine differences
between apoptotic and necrotic cells

-
Alanine and glutamate
could be novel biomarkers of apoptosis

- We have further demonstrated in this study that
this assay could be adapted easily for monitoring the effects of anticancer drugs in cell culture
: The adapted NBS assay has strong potential as a tool in preclinical testing of cancer drug efficacy
Alanine
Glutamate
HALLMARKS
Cell viability and apoptosis validadtion after etoposide or 5-Fu treatment
Main objective:
to better understand and determine specific biomarkers for the process of apoptosis
Metabolomic signatures of etoposide or 5-Fu-treated cells
Amino acids and acylcarnitines were quantified in three groups of cells:


-
Control

(DMSO, 100% viability)
-
Apoptotic

(etoposide or 5-Fu)
-

Necrotic
(DMSO + heat)

In this experimental setup the metabolite levels of apoptotic cells were compared with those of control and necrotic cells

11 of the 42 analyzed metabolites were significantly regulated in apoptosis and/or necrotic-induced cell lines
Alanine and glutamate levels indicate apoptotic cells regardless inducing drugs
7 metabolites were found to be specific for apoptotic processes

After the new results of previous and present studies, only alanine and glutamate could be considered useful markers of apoptosis
Full transcript