enbioq 2014 rodrigues
TRANSCRIPT
• Approximately 600 and 400 spots were found in 2D gels run in the pH 4-7 and 6-11 ranges respectively, and stained with Colloidal Coomassie Blue. Currently, a comparative analysis of the protein profile is underway and will later be statistically validated.
• Future work: identification of differentially expressed proteins present in samples and controls by mass spectrometry techniques; optimization of the cardiomyocytes extraction process; validation of the cardiotoxicity biomarkers.
V.M. Costa, F. Carvalho, J.A. Duarte, M.D.L. Bastos, F. Remião, The heart as a target for xenobiotic toxicity: the cardiac susceptibility to oxidative stress., Chem. Res. Toxicol. 26 (2013) 1285–311.
This work was supported by Fundação para a Ciência e Tecnologia (FCT) - project [EXPL/DTP-FTO/0290/2012] – QREN’s iniciative funded by EU/FEDER through COMPETE - Operational Programme for Competitiveness Factors.
HL-1
10 min 5000 rpm
4°C
Culture Incubation (MTX 1µM and 10µM, for
12h)
Harvest (Scropping PBS + Ca2+ + Mg2+)
Cell Lysis (7M Urea, 2M Thiourea,
4%CHAPS, 0.5% IPG Buffer)
Supernatant Sonicate 15 x 2 s bursts
Pellet (Membrane proteins)
Supernatant 12 min 12000 rpm
4°C
Pellet
Supernatant (Proteins)
Bradford Quantification IEF SDS-PAGE
Pellet
A
B
A
pH
MW
B
C D
Figure 2 – 2-D gel images of the control (A and B), 10 µM MTX incubation for 12h (C and D), Proteins (150 µg) from cell extracts of HL-1 cardiomyocytes were separated on a pH 4–7 strip (A and C) and pH 6-11 strip (B and D), followed by SDS-PAGE on a 12.5% acrylamide gel. Proteins were visualized by colloidal coomassie blue staining. The image gels were analyzed by Image Master Workstation software (Nonlinear).
Figure 1 – A: HL-1 cardiomyocytes control; B: HL-1 cardiomyocytes with 10 µM MTX incubation for 12h. Cell cultures were assessed morphologically by phase contrast microscopy at the selected time-points (microphotographs were taken with a Nikon Eclipse TS100 equipped with a Nikon DS-Fi1 camera).
Mitoxantrone (MTX) , a synthe3c anthracenedione, has been used as an an3carcinogen drug in various types of cancer cancer (prostate, metastasized breast cancer, lymphoma, acute myeloid leukemia) as well as in mul3ple sclerosis. This molecule has the ability to inhibit type II topoisomerase preven3ng DNA linkage and consequently the progression of the cellular cycle. Studies have found that the use of this drug has cardiotoxic effects that cause heart failurePrevious studies have found that the use of MTX has cardiotoxic effects causing heart failure and leading to an increased risk of death. The mechanisms underlying the damage caused by MTX to the cardiac muscle are being studied using proteomic tools (2D electrophoresis, 2DE). This approach will enable the iden3fica3on of novel biomarkers of cardiac toxicity. The HL-‐1 cell line used in this work possesses similar characteris3cs to human cardiomyocytes.
4 7 6 11
Co
ntro
l 10
µM
MTX