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