hyl dox ncsas poster 16.3.27 r-zj (2)
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Results
Abstract
Discussion & Summary
I. Doxorubicin reduces the presence of intracellular glutathione, an
indicative of oxidative stress.
II. Doxorubicin induces increased gene expressions for intracellular
adhesion molecules (ICAM, VCAM, E-selectin) that are key
components of beginning stages of inflammation.
III. The proposed study may lay a foundation for further investigation
of Dox-induced oxidative stress on cardiomyopathy of H9c2
cardiomyocytes.
DOXORUBICIN INCREASES THE EXPRESSION OF ADHESION MOLECULES AND MODIFIES
THE STATUS OF INTRACELLULAR GLUTATHIONE IN RAT H9C2 MYOCARDIAL CELLS
Ho Young Lee1, Halley Shah1, Rojin Chitrakar1, McKenna Kormanik1, Hong Zhu2, Robert Y. Li1,2 , Zhenquan Jia1
1Department of Biology, University of North Carolina at Greensboro, NC
2Campbell University School of Osteopathic Medicine, Buies Creek, NC
Materials and Methods
Doxorubicin (Dox) is one of the most effective anticancer drugs. The
downside associated with the use of Dox is the high risk of irreversible
cardiomyopathy and cardiotoxicity, but the precise mechanisms remain
to be defined. In this study, we used qRT-PCR to examine the expression
of vascular adhesion molecule-1 (VCAM-1), intercellular adhesion
molecule-1 (ICAM-1) and E-Selectin following Dox treatment in rat H9C2
cardiomyocytes. Our results showed that Dox, at a clinically relevant
plasma concentration, significantly increased the expression of adhesion
molecule VCAM-1, ICAM-1, and E-Selectin in rat H9c2 cardiomyocytes.
Further, Dox also changed the status of intracellular glutathione, an
indicative of oxidative stress. These results provide the direct evidence
that the expression of adhesion molecules and oxidative stress could be
a critical player in Dox-induced cardiomyopathy and cardiotoxicity.
Doxorubicin (Dox) is an antitumor anthracycline antibiotic, which
intercalates into DNA resulting in inhibition of DNA replication,
and protein synthesis.
Introduction & Hypothesis
Dox-induced cardiomyopathy can cause congestive heart failure
with mortality rate of as high as 50%, especially when cumulative
doses exceeding 500 mg/m2.
10%~25% of Dox treatment patients develop cardiotoxicity. While
most suffer from cardiotoxicity during chemotherapy, some even
suffer after completion of chemotherapy.
This study examined the effect of widely used anti-cancer drug
doxorubicin on H9c2 cells. We propose that Dox promotes
oxidative stress in cardiomyocytes leading to cellular depletion of
antioxidants such as glutathione, ultimately resulting in
cardiomyopathy.
Cell culture and harvesting: H9c2 cells were cultured in DMEM 10%
fetal bovine serum (FBS) and 1% 10,000 U/ml penicillin 10mg/ml
streptomycin in 75cm2 flasks with filtered tops. Media was changed on
alternating days. Incubator was set on 37°C with 5% CO2. H9c2 cells
were washed with sterile PBS before harvesting. Cells were
trypsinized at 80% confluence following centrifugation at 1000 rpm for 7-8 minutes and resuspension in DMEM
media.
Glutathione assay: H9c2 cells, 80% confluent, were exposed to Dox for 24 hours. 12.5 μl of 25% meta-phosphoric
acid and 0.1% sodium phosphate buffer at pH 8.0 were added to cell lysate followed by 10-minute incubation at 4º C.
The sample was centrifuged at 13,000 rpm for 5 minutes, and 10 μl of the resulting supernatant was further incubated
with 0.1% OPT in methanol and 0.1% sodium phosphate buffer for 15 minutes at room temperature in the absence of
light. Fluorescence intensity of GSH-OPT was measured by excitation at 350 nm and emission at 420 nm. The sample
GSH content was calculated using a GSH standard curve and was expressed as percentage compared to an untreated
control.
NQO1 assay: H9c2 cells, 80% confluent, were exposed to Dox for 24 hours. NQO1 reaction mix was prepared with
12mL of 50mM Tris-HCl, pH7.5, 0.08% Triton X-100, 36uL of 50mM NADPH, and 48uL of 20mM DCPIP. The cell lysate
was mixed with Tris-HCl buffer at pH 7.5, NADPH, and Dichlorophenolindophenol (DCPIP). The enzyme activity was
determined by following the continuous reduction of DCPIP which leads to decreased absorbance at 600 nm.
qRT-PCR: H9c2 cells were exposed to Dox for 3 hours at 80% confluence. Total RNA from the cells was extracted
using the Trizol Reagent. The isolated RNA was then reverse transcribed to cDNA. One μl of the amplified cDNA was
added to the SYBR® Green real-time PCR master mix along with specific primer sets (ICAM F&R, VCAM F&R, E-
Selectin F&R). The reaction cycles were performed in the Applied Biosystems thermal cycler. A total of 40 cycles was
performed for each run as following: 95ºC for 15 seconds, 61ºC for 30 seconds, and 72ºC for 30 seconds. Expression
of the housekeeping gene, GAPDH, was also acquired to normalize the expression of other target genes. Quantification
of gene expression was determined using the comparative threshold cycle CT method and expressed as fold-change
compared to the untreated control.
References
• Chatterjee, K., Zhang, J., Honbo, N., & Karliner, J. S. (2009). Doxorubicin Cardiomyopathy.
Cardiology, 155-162.
• Zhang, S., Liu, X., Bawa-khalfe, T., Lu, L., Lyu, Y. L., Liu, L. F., & Yeh, E. T. H. (November
01, 2012). Identification of the molecular basis of doxorubicin-induced cardiotoxicity. Nature
Medicine, 18, 11, 1639-42.
• Singh, P., Sharma, R., McElhanon, K., Allen, C. D., Megyesi, J. K., Benes, H., & Singh, S. P.
(January 01, 2015). Sulforaphane protects the heart from doxorubicin-induced toxicity. Free
Radical Biology & Medicine, 86, 90-101.
C B A
Fig 1: Rat H9c2 cardiomyocytes were treated with or without the indicated concentration of doxorubicin
(Dox) for 3 hours. The mRNA levels of (A) vascular adhesion molecule-1 (VCAM-1), (B) intercellular adhesion
molecule-1 (ICAM-1), and (C) intercellular adhesion molecule E-Selectin were measured by qRT-PCR and
normalized to GAPDH expression. Data are mean ± SEM. *P<0.05 compared to control group
B A
Figure 2: Dox decreased cellular GSH and NQO1 in cardiomyocytes. H9c2 cells were treated with various
concentrations of Dox for 24 hours and (A) cellular GSH content was measured fluorometrically with OPT. (B) NQO1
activity was measured by following the continuous reduction of DCPIP at 600nm. Data are mean ± SEM. *P<0.05
compared to control group (n=3).
Fig. 4. Effects of D3T pretreatment on doxorubicin-mediated cytotoxicity
in cardiomyocytes. Cells were pretreated with the indicated concentrations
of D3T for 24 h, followed by incubation with various concentrations of
doxorubicin for another 48 h. Cell viability was determined using MTT
reduction assay. * significantly different from control; # significantly different
from 10 µM D3T; & significantly different from 25 µM D3T.
ROS-induced
cardiomyocyte injury?
Left Ventricle
Low output Normal output
Cardiomyopathy
Prolonged Exposure to
Dox
↑ Oxidative stress ↑ Inflammation
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