JAK2/STAT3 Pathway Mediates Protection of Metallothionein Against Doxorubicin-Induced Cytotoxicity in Mouse Cardiomyocytes

Abstract

Doxorubicin (Dox) is one of the most important anticancer agents; however, its clinical application is limited by its severe cardiotoxicity. In our previous study, we found that the gene expression levels of the Janus-activated kinase/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway were different between MT^−/− cardiomyocytes and MT^+/+ cardiomyocytes when they were treated with Dox. Thus, this study was intended to investigate the role of JAK2/STAT3 pathway in metallothionein (MT) protection of Dox-induced cardiotoxicity. Tyrphostin AG490 (α-cyano-(3,4-dihydroxy)-N-benzylcinnamide) is a synthetic protein tyrosine kinase inhibitor which at first has been considered as a specific JAK2 inhibitor and can inhibit the JAK2/STAT3 signaling pathway. In the present study, AG490 was used to assess the role of JAK2/STAT3 in MT protection against Dox-induced cardiotoxicity. The AG490 can attenuate the MT protection by increasing lactate dehydrogenase and the number of apoptotic cells. Interestingly, pretreated with AG490, MT^−/− cardiomyocytes were more sensitive than MT^+/+ to Dox-induced cytotoxicity as measured by reactive oxygen species generation, lipid peroxidation, and protein carbonylation. Metallothionein 1 and MT-2 messenger RNA were upregulated by Dox, and AG490 decreased the protein expression of MT-1 and MT-2. After Dox treatment, the protein expression of p-Jak2 and p-Stat3 levels was significantly increased in MT^+/+ cardiomyocytes, suggesting that the JAK2/STAT3 pathway was partially involved in MT protection against Dox-induced cardiotoxicity.

Introduction

Doxorubicin (Dox), an anthracycline antibiotic, is one of the most important and widely used anticancer agents. ¹ It has been a crucial agent to treat acute leukemia, breast cancer, and Hodgkin and non-Hodgkin lymphoma. ² However, it has severe cardiotoxicity, which is a major barrier to its clinical application. ³ Several proposed mechanisms are thought to be involved in Dox-induced heart failure, including DNA intercalation, free radical generation, and damage to cell membranes. Most studies support this opinion that oxidative stress plays a significant role in the pathogenesis of Dox-induced cardiotoxicity. ⁴

Metallothionein (MT) is a low-molecular-weight, thiol-rich protein which functions by scavenging free radicals. ⁵ As for its 4 isoforms, MT-I and MT-II appear in all tissues and organs in mammals, MT-III is brain specific, and MT-IV is only present in some tissues containing stratified squamous epithelial cells. ^{6 –8} Metallothionein antioxidant property was first explored extensively in the early 1980s. And, there is clear evidence that MT can scavenge reactive oxygen species (ROS), either from toxic metals or from other chemicals, so MT can alleviate tissue damage induced by oxidative stress. ⁹ In our previous studies, MT has been demonstrated to be an effective protector against Dox-induced cardiomyopathy, but the exact mechanisms have not been fully elucidated. ^10,11

The Janus-activated kinase/signal transducer and activator of transcription (JAK-STAT) pathway directly transmits transcriptional information with transmembrane receptors to targeted gene acceptors present in the nucleus, providing a mechanism for transcriptional regulation without secondary messengers. ¹² The JAK proteins are cytosolic tyrosine kinases and include 4 family members: JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2). ¹³ Janus-activated kinase 1 and JAK2 are expressed in most tissues, whereas JAK3 and TYK2 are mainly expressed in hematopoietic cells and certain carcinoma cells. ¹⁴ The STAT proteins have 7 subtypes distributed in mammalian cells: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6. ¹³ Signal transducer and activator of transcription 3 is overexpressed in the heart. The JAK-STAT pathway can be activated by various cytokines and growth factors, such as the interleukin (IL)-6 and IL-10 family of cytokines. ¹⁵ Extensive research on the heart have indicated that the JAK-STAT pathway plays a critical role in apoptosis, heart transplant, cardiac hypertrophy, oxidative stress, and ischemia–reperfusion injury. ^16,17 In addition, a previous study showed that overexpression of STAT3 in the heart can protect against Dox-induced cardiomyopathy. ¹⁸ Some studies also demonstrated that activation of STAT3 protein can protect myocardium from ischemia–reperfusion injury in vivo, partially through MT1 and MT2. ¹⁹

In previous experiments in our laboratory, we found that the gene expression profile of the JAK-STAT pathway increased in wild-type mice (MT^+/+) after being given Dox treatment. We thus hypothesize that MT may prevent Dox-induced cardiotoxicity involving the JAK-STAT pathway. Therefore, the objective of this study was to investigate the role of the JAK-STAT pathway in MT protection of Dox-induced cardiotoxicity.

Materials and Methods

Chemicals and Antibodies

Doxorubicin hydrochloride, AG490, and bromodeoxyuridine (Brdu) were obtained from Sigma Aldrich (St. Louis, MO, USA). Dulbecco modified Eagle medium (DMEM) was obtained from Gibco (USA). Fetal bovine serum (FBS) was provided by Hyclone (Australia). The Hoechst 33258 staining kit was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Lactate dehydrogenase (LDH) assay kit was provided by Biosino Bio-Technology and Science Inc (Beijing, China). Rabbit monoclonal anti–p-Stat3 (tyr-705) antibody, rabbit monoclonal anti-Stat3 (AB-727) antibody, rabbit monoclonal anti–p-Jak2 (tyr-1007) antibody, rabbit monoclonal anti-Jak2 (AB-1007) antibody, mouse anti-MT, and goat anti-rabbit immunoglobulin (Ig) G–horseradish peroxidase (HRP) were obtained from Sigma Aldrich (St. Louis, MO, USA). Mouse monoclonal anti–β-actin and goat anti-mouse IgG-HRP were purchased from Zhongshan Goldenbridge Biotechnology (Jiangsu, China).

Animals

The Murdoch Institute of the Royal Children’s Hospital (Parkville, Australia) provided the homozygous wild-type mice (MT^+/+). Metallothionein-null mice (MT^−/−) were deficient both in MT-I and MT-II genes. All animals were maintained at a controlled temperature of 23°C ± 1°C and in a 12-hour light/dark cycle in ventilated animal rooms. All the animal experiments were carried out after being approved by the Institutional Animal Care and Use Committee and were in conformity with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

Cardiomyocyte Culture

Primary cultures of cardiac ventricular myocytes were prepared as described previously with minor modifications. ²⁰ Briefly, hearts from 1- to 3-day-old MT-I/II knockout (MT^−/−) or wild-type (MT^+/+) mice were removed aseptically and kept in ice-cold DMEM. The hearts were washed 3 times with the same DMEM and minced into small fragments. The cells were dissociated at 37°C with collagenase (0.1% wt/vol) and trypsin (0.05% wt/vol). Cardiac cells released from digestion were added to an equal volume of DMEM supplemented with 5% FBS until all the cardiac cells were isolated. This final mixture was then centrifuged for 10 minutes at 1000 rpm, and the cells were resuspended in the FBS-DMEM (DMEM supplemented with 15% FBS [vol/vol], 80 U/mL penicillin, and 100 U/mL streptomycin). These myocytes were then collected and plated in 35-mm culture dishes, 25-cm² culture flasks, or 96-well culture plates at a density of 1.0 × 10⁵ cells/mL. In order to inhibit fibroblast growth, Brdu was added to the medium after 48 hours.

Drug Treatment

Doxorubicin was dissolved at 1 mmol/L in deionized water, and further dilutions were made with culture medium. Dox was used at a final concentration of 2 μmol/L. AG490, a specific inhibitor of JAK2/STAT3 signaling, which has been used in other studies, ^21,22 was utilized in our study. AG490 was dissolved at 100 mmol/L in dimethylsulfoxide (DMSO) and at a final concentration of 0.1% DMSO in culture medium. Cells were then treated with AG490 60 minutes prior to Dox treatment at a final concentration of 5 μmol/L. During Dox or AG490 exposure, the FBS-DMEM was removed and replaced with fresh serum-free DMEM.

RNA Extraction and Reverse Transcription–Polymerase Chain Reaction

Six-day-old MT^+/+ cardiomyocytes were treated with AG490 (5 μmol/L) alone, Dox (2 μmol/L) alone, AG490 (5 μmol/L) + Dox (2 μmol/L), or diluent (control). After 24 hours, total RNA was harvested using Trizol reagent (Invitrogen, USA) on the basis of the manufacturer’s instructions. Complementary DNA synthesis and the analysis of the results were obtained according to a previous study. ²³ The sense and antisense primer sequences used were MT-1:5′-AAGAGTGAGTTGGGACACCTT-3′, 5′-CGAGACAATACAATGGCCTCC-3′; and MT-2:5′-GCCTGCAAATGCAAACAATGC-3′,5′-AGCTGCACTTGTCGGAGC-3′. The polymerase chain reaction (PCR) program was as follows: denaturation at 95°C for 5 minutes, 30 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 25 seconds, followed by a 72°C elongation step for 10 minutes. Twelve microliters of each PCR product was resolved by 2% (wt/vol) agarose gel electrophoresis.

Western Blot Analysis

Cardiac cells were incubated in 25-cm² culture flasks. Nuclear and cytoplasmic proteins were extracted using cell lysis buffer which contained protease inhibitors (1 mmol/L of phenylmethanesulfonyl fluoride) and 1% phosphatase inhibitors. The concentrations of these proteins were determined according to the bicinchoninic acid (BCA) protein assay. An equal amount of protein (20 μg) was loaded for 1-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) in a discontinuous system using 12% separating gel and 5% stacking gel. Samples were separated by SDS-PAGE electrophoresis and transferred onto polyvinylidene fluoride membranes. Membranes were blocked in blocking buffer (Tris-buffered saline, 5% nonfat dried milk, and 0.1% Tween (Amresco, USA)) for 2 hours, followed by incubation with primary antibodies (mouse anti-MT, 1:1,000; rabbit anti–p-Stat3, 1:1,000; rabbit anti-Stat3, 1:1,000; rabbit anti-p-Jak2, 1:1,000; rabbit anti-Jak2, 1:1,000; or mouse anti–β-actin, 1:2,000) at 4°C overnight. Following 3 washes in Tris-buffered saline and Tween (TBST) for 10 minutes at room temperature, the membranes were incubated with secondary antibodies (goat anti-rabbit 1:2,000 and goat anti-mouse 1:5,000) in 5% nonfat milk (diluted in TBST) for 45 minutes while shaking at room temperature and washed in TBST for 10 minutes 3 times. Membranes were then exposed to enhanced chemiluminescence film for detection. Band density was quantified using a Quantity One image densitometer (Bio-Rad, USA).

Morphological Observation

Morphological characteristics of the cultured cardiomyocytes were observed with an inverted microscope (Olympus, Japan). On the sixth day of culture, the cultured cardiomyocytes were treated with AG490 (5 μmol/L) alone, Dox (2 μmol/L) alone, AG490 (5 μmol/L) + Dox (2 μmol/L), or diluent (control) for 24 hours.

Determination of Apoptotic Cells

Apoptosis in cardiomyocytes was detected via Hoechst 33258 staining. Six-day-old MT^+/+ and MT^−/− cardiomyocytes were treated with AG490 (5 μmol/L) alone, Dox (2 μmol/L) alone, AG490 (5 μmol/L) + Dox (2 μmol/L), or diluent (control) for 24 hours. After being washed twice with cold phosphate-buffered saline (PBS) and fixed in 4% formaldehyde for 10 minutes, the cultured cells were incubated with Hoechst 33258 (5 μg/mL) for 10 minutes at room temperature in the dark. Subsequently, the cells were washed again with cold PBS. Finally, cell nuclei analyses were conducted by an inverted fluorescence microscope (DMI3000 B; Leica Microsystems Ltd (Germany)) with excitation at 350 nm and emission at 460 nm. The number of apoptotic nuclei was measured on at least 6 randomly selected areas from 3 coverslips of each group. The percentage of apoptotic cells relative to the total number of cells was used to express the results.

Lactate Dehydrogenase Release

Six-day-old MT^+/+ and MT^−/− cardiomyocytes were treated with AG490 (5 μmol/L) alone, Dox (2 μmol/L) alone, AG490 (5 μmol/L) + Dox (2 μmol/L), or diluent (control). After 24 hours, the supernatants from the culture media were collected. Lactate dehydrogenase activity was measured using a commercial LDH assay kit as per the manufacturer’s instructions. Absorbance at 340 nm was measured by a microplate reader. The data were presented as the percentage of the total releasable LDH.

Analysis of ROS

Levels of oxidative stress were analyzed by the measurement of ROS. The determination of intracellular ROS production was on the basis of the oxidation of 2′,7′-dichlorodihydrofluorescein to fluorescent 2′,7′-dichlorofluorescein (DCF) as described previously. ²⁴ Following exposure to AG490 (5 μmol/L) alone, Dox (2 μmol/L) alone, AG490 (5 μmol/L) + Dox (2 μmol/L), or diluent (control) for 24 hours, the cultured cells were washed with PBS and incubated with a 4 μmol/L solution of fluorescent probe (2′,7′-dichlorodihydrofluorescein diacetate [H₂-DCFDA]) dissolved in culture medium at 37°C for 40 minutes. Having been washed 3 times with cold PBS, the cells were visualized with an inverted fluorescence microscope (DMI3000 B; Leica Microsystems Ltd) with excitation at 488 nm and emission at 530 nm. The captured images were processed and quantified by Image-Pro Plus 6.0 software.

Lipid Peroxidation Assay

The thiobarbituric acid reactive substances (TBARS) method was used to determine the effect of Dox-induced lipid peroxidation according to a previously reported method with slight modification. ²⁵ After treatment by AG490 or Dox, nuclear and cytoplasmic proteins were extracted as described previously, and protein concentrations were measured according to a BCA protein assay. Two hundred microliters of cell lysates was added to 200 μL 20% trichloroacetic acid (TCA) and 100 μL 1% 2-thiobarbituric acid. After being placed in a water bath for 45 minutes, n-butanol was added to the mixture. The reaction mixture was centrifuged at 3000 rpm for 10 minutes and determined at 532 nm. The TBARS levels were calculated from a standard curve and normalized to the protein content.

Analysis of Protein Carbonyl Content

As lipid peroxidation, protein carbonylation is also a marker of oxidative stress. These samples were incubated with an equal volume of 0.1% (wt/vol) 2,4-dinitrophenylhydrazine (DNPH) in 2 N HCl for 1 hour at room temperature in the dark and then treated with 20% TCA. After centrifugation, the precipitate was extracted 3 times with ethyl alcohol/ethyl acetate (EtOH/EtOAc) and dissolved in 8 mol/L guanidine hydrochloride. The absorbance was recorded at 370 nm. The carbonyl concentration was based on the molar extinction coefficient of 22.0 mmol/(L·cm). The results were expressed as µmol of DNPH incorporated/g protein.

Statistical Analysis

All data were presented as mean ± standard deviation (SD) and analyzed by 1-way analysis of variance, followed by least significant difference post hoc test using the SPSS 13.0 statistical package. Differences between MT^+/+ controls and MT^−/− mice cardiomyocytes were analyzed by t test. The criterion for statistical significant was P < 0.05.

Results

Effects of Dox and AG490 on MT-1, MT-2 Messenger RNA, and Protein Expression

As shown in Figure 1, the messenger RNA (mRNA) levels of MT-1 and MT-2 were assayed by reverse transcription–polymerase chain reaction (Figure 1A), and the protein expression was assayed by Western blot analysis (Figure 1B). The results indicated that MT-1 and MT-2 mRNA were upregulated, and the protein expression of MT was significantly increased by Dox. In contrast, the increased MT expression in the Dox + AG490 group was blocked by 5 μmol/L AG490.

Figure 1.

AG490 attenuates doxorubicin (DOX)-induced enhancement of expression of metallothionein (MT)-1 and MT-2 messenger RNA (mRNA) and protein in MT^+/+ cardiomyocytes. Six-day-old MT^+/+ cardiomyocytes were treated with AG490 (5 μmol/L) alone, Dox (2 μmol/L) alone, AG490 (5 μmol/L) + Dox (2 μmol/L), or diluent (control) for 24 hours. Representative image of MT-1 and MT-2 mRNA and quantitative analysis of the expression of MT-1 and MT-2 mRNA (A). Representative Western blots of MT-I/II in MT^+/+ cardiomyocytes and quantitative analysis of the protein expression of MT-I/II (B). Data are expressed as mean ± standard deviation (SD; n = 3). *P < 0.05 significantly different from control values; ^# P < 0.05 significantly different from the Dox-treated MT^+/+ cardiomyocytes.

Effects of MT on JAK/STAT Phosphorylations

MT^+/+ cardiomyocytes were exposed to 2 μmol/L Dox alone for 24 hours. The phosphorylation of JAK2 and STAT3 (Figure 2) was examined by Western blot analysis. In the absence of Dox, the JAK2 and STAT3 phosphorylation was at a low level. After 2 μmol/L Dox administration, the phosphorylation of JAK2 and STAT3 was enhanced.

Figure 2.

Doxorubicin (DOX) induces tyrosine phosphorylation of Janus-activated kinase (JAK2) and signal transducer and activator of transcription 3 (STAT3) after treatment with 2 μmol/L Dox in mouse cardiomyocytes overexpressing metallothionein (MT^+/+; metallothionein [MT]-1 and MT-2). Identical results were obtained from 3 independent experiments, whereas the representative blots only shown the results from 1 experiment (A). Quantitative analysis of the protein expression of p-JAK2/JAK2 and p-STAT3/STAT3 (B).

Morphological Observations of MT^+/+ and MT^−/− Cardiomyocytes

MT^+/+ and MT^−/− cardiomyocytes were isolated and cultured separately. As shown in Figure 3, there was no obvious difference between MT^+/+ and MT^−/− cardiomyocytes as evaluated by cell morphology on the sixth day after culture. After treatment with 2 μmol/L Dox for 24 hours, the morphology of cells changed dramatically. However, MT^−/− cardiomyocytes showed a remarkably slow rate of spontaneous transformation and exhibited minimal pseudopodia and intermediate vacuolization in comparison with MT^+/+ cardiomyocytes. When both types of cardiomyocytes were pretreated for 60 minutes with 5 μmol/L AG490, the selective Jak2 inhibitor, and then exposed to Dox for 24 hours, the MT^+/+ and MT^−/− cardiomyocytes aggregated and formed a ball-like appearance compared with Dox treatment alone. The transformation was more obvious in MT^−/− cardiomyocytes.

Figure 3.

Morphological observations of MT^+/+ and MT^−/− cardiomyocytes. No obvious difference was found between untreated MT^+/+ and MT^−/− cardiomyocytes. MT^−/− cardiomyocytes exhibited minimal pseudopodia and intermediate vacuolization after treatment with 2 μmol/L doxorubicin (Dox) alone for 24 hours. Compared with Dox treatment, MT^+/+ cardiomyocytes showed appearance of minimal pseudopodia and intermediate vacuolization after pretreatment with 5 μmol/L AG490 for 60 minutes followed by cotreatment with 2 μmol/L Dox for 24 hours (original magnification, ×400).

Metallothionein Deficiency Exacerbates Dox-Induced Cytotoxicity

The Dox-induced cytotoxicity in the primary cultures of MT^+/+ and MT^−/− cardiomyocytes was measured by LDH leakage and apoptosis. The release of the cytosolic enzyme LDH from cardiomyocytes is commonly used as a measurement of Dox and other drug-induced damage. ²⁶ No difference in LDH release was detected in cultures without any treatment. After 2 μmol/L Dox treatment, the significant increase in the release of LDH leakage from MT^−/− cardiomyocytes was reported (shown in Figure 4) in comparison to MT^+/+ cardiomyocytes. After these cells were pretreated with 5 μmol/L AG490, the LDH release increased in MT^+/+ cardiomyocytes and MT^−/− cardiomyocytes compared with the Dox-alone group, but the increase was very minor in MT^−/− cardiomyocytes.

Figure 4.

Janus activated kinase 2 (JAK2) inhibition aggravated LDH release in MT^+/+ and MT^−/− cardiomyocytes exposed to 2 μmol/L Dox and 5 μmol/L AG490 for 24 hours. AG490 is an inhibitor of JAK2. Data are expressed as the mean ± standard deviation (SD; n = 10). *P < 0.05 significantly different from control values; 0P < 0.05 significantly different from MT^+/+ cardiomyocytes for the same treatments; ^# P < 0.05 significantly different from the Dox-treated MT^+/+ and MT^−/− cardiomyocytes.

Apoptosis is a critical cellular process involved in Dox-induced cytotoxicity. Doxorubicin has been shown to induce apoptosis both in vitro and in vivo. ¹⁰ In order to test this, cells were stained using Hoechst 33258. As shown in Figure 5, normal cells were seen as round-shaped nuclei with a homogeneous fluorescence intensity. There was no significant difference between the untreated MT^+/+ and MT^−/− cardiomyocytes, with the percentage of apoptotic cells at 15.1% and 16.4%, respectively. Doxorubicin treatment induced significant changes in the nuclear morphology, with an apoptotic level of 44.3% in MT^+/+ cardiomyocytes and 72.2% in MT^−/− cardiomyocytes. But, there was no significant difference in cultures exposed to 5 μmol/L AG490 alone. The data represented in Figure 5B demonstrated that added AG490 almost doubled the number of apoptotic nuclei in MT^+/+ cardiomyocytes compared with the Dox-alone group, but such a change was not observed in MT^−/− cardiomyocytes.

Figure 5.

Janus activated kinase 2 (JAK2) inhibition induced the cell apoptosis seriously in MT^+/+ cardiomyocytes. A, Morphology of apoptotic cells, representative fluorescent microscopic images of cardiomyocytes. Cell nuclei were observed with magnification at ×40 and monitored with excitation at 350 nm and emission at 460 nm. B, Percentage of apoptotic cells in MT^+/+ and MT^−/− cardiomyocytes. Data are expressed as the mean ± standard deviation (SD; n = 3). *P < 0.05 significantly different from control values; 0P < 0.05 significantly different from MT^+/+ cardiomyocytes for the same treatments; ^# P < 0.05 significantly different from the Dox-treated MT^+/+ and MT^−/− cardiomyocytes.

Oxidative Stress Studies

Oxidative stress was monitored in terms of production of ROS, as Dox has been known to generate ROS during its metabolism in the heart. ⁵ 2′,7′-Dichlorodihydrofluorescein diacetate can be used as a fluorescent probe and is a reporter of single cellular ROS generation. Figure 6A is a representative picture of the DCF fluorescence in cardiomyocytes during various treatments. These images were digitized and analyzed for ROS-induced DCF fluorescence (Figure 6B). MT^+/+ and MT^−/− cardiomyocytes were treated with 2 μmol/L Dox for 24 hours, and Dox caused 14-fold and 22-fold increases in ROS, respectively. In the AG490-pretreated cultured cardiomyocytes, there was a 1.5-fold increase in MT^+/+ cardiomyocytes in comparison with the Dox-alone group, whereas no obvious increase was found in MT^−/− cardiomyocytes.

Figure 6.

Janus activated kinase 2 (JAK2) inhibition increased ROS generation in MT^+/+ cardiomyocytes. A, Representative fluorescent microscopic images of MT^+/+ and MT^−/− cardiomyocytes. Reactive oxygen species (ROS) generation was measured by 2′,7′-dichlorodihydrofluorescein diacetate (H₂-DCFDA) staining. The cells were visualized with magnification at ×40 and monitored with excitation at 488 nm and emission at 530 nm. B, Results for different treatment groups were normalized by mean fluorescence intensity. Data are expressed as mean ± standard deviation (SD; n = 10). *P < 0.05 significantly different from control values; 0P < 0.05 significantly different from MT^+/+ cardiomyocytes for the same treatments; ^# P < 0.05 significantly different from the Dox-treated MT^+/+ and MT^−/− cardiomyocytes.

Lipid Peroxidation

Lipid peroxidation can be measured by a thiobarbituric acid reactive substances (TBARS) assay. Doxorubicin can initiate lipid peroxidation and give rise to maximum TBARS formation. ²⁷ The data in Figure 7 show that the level of the lipid peroxidation products was elevated by Dox in the 2 types of cardiomyocytes. However, this effect was more remarkable in MT^−/− cardiomyocytes. After both AG490 and Dox treatment, TBARS were found to be dramatically increased in MT^+/+ cardiomyocytes compared to Dox alone, which was not dramatic in MT^−/− cardiomyocytes.

Figure 7.

The doxorubicin (Dox)-induced lipid peroxidation aggravated when Janus activated kinase 2 (JAK2) was blocked in MT^+/+ cardiomyocytes. Lipid peroxidation was measured by a thiobarbituric acid reactive substances (TBARS) assay. The content of methane dicarboxylic aldehyde (MDA) was measured by TBARS assay at 532 nm. Data are expressed as mean ± standard deviation (SD; n = 3). *P < 0.05 significantly different from control values; 0P < 0.05 significantly different from MT^+/+ cardiomyocytes for the same treatments; ^# P < 0.05 significantly different from the Dox-treated MT^+/+ and MT^−/− cardiomyocytes.

Protein Carbonylation

2,4-Dinitrophenylhydrazine assay is a classic method to measure the protein carbonyl content. As seen in Figure 8, the protein carbonyl content in MT^+/+ and MT^−/− cardiomyocytes (4.86 and 4.99, respectively) did not show significant differences from the control group. An about 2.8-fold and 4-fold increases (13.83 and 20.24, respectively) were seen in MT^+/+ and MT^−/− cardiomyocytes, respectively, exposed to 2 μmol/L Dox for 24 hours. As compared with the Dox-alone group, the protein carbonyl content was increased by as much as 2-fold and 1.5-fold (28.91 and 30.52, respectively) in MT^+/+ and MT^−/− cardiomyocytes treated with Dox and AG490, respectively.

Figure 8.

Doxorubicin (DOX)-induced protein oxidation and the effect of AG490. Carbonyl content is a marker of protein oxidation and is detected by 2,4-dinitrophenylhydrazine (DNPH) assay. Data are expressed as mean ± standard deviation (SD; n = 3). *P < 0.05 significantly different from control values; 0P < 0.05 significantly different from MT^+/+ cardiomyocytes for the same treatments; ^# P < 0.05 significantly different from the Dox-treated MT^+/+ and MT^−/− cardiomyocytes.

Discussion

To test our hypothesis that JAK2/STAT3 pathway participated in MT protection against Dox-induced cardiotoxicity, we established a primary cardiomyocytes culture system from MT^−/− and MT^+/+ mice and used the inhibitor AG490, which is selective for JAK2. The test results show that MT-1 and MT-2 mRNA were upregulated by Dox, but AG490 can prevent this upregulated expression of MT-1 and MT-2. Western blot analysis indicated that the protein expression of MT was significantly elevated and that Jak2 and Stat3 were phosphorylated in MT^+/+ cardiomyocytes upon Dox treatment. However, this increased MT expression was blocked by AG490. Consequently, AG490 can suppress the MT protection against Dox-induced toxicity in MT^+/+ cardiomyocytes. However, this suppression was not apparent in MT^−/− cardiomyocytes. Thus, this study provides evidence that MT may prevent Dox-induced cardiotoxicity involving the JAK2/STAT3 pathway.

Doxorubicin is an effective anticancer agent used to treat many human neoplasms, including malignant lymphomas and acute leukemias as well as a number of solid tumors. However, its serious cardiotoxicity limits its clinical application. ³ Previous research have shown that MT which serves as a sacrificial scavenger of free radicals can provide protection against Dox-induced cardiotoxicity. However, the exact mechanisms have not been clearly elucidated. ^10,11 In our previous studies, we found that MT can inhibit Dox-induced ROS generation, increasing levels of lipid peroxidation and protein carbonylation in MT^+/+ cardiomyocytes. In addition, MT^−/− deficiency showed higher levels of cytotoxicity. In the present study, we also found that Dox induced significant cardiac morphological changes and increased LDH leakage and apoptotic cells in MT^−/− cardiomyocytes over MT^+/+ cardiomyocytes. These observations are consistent with the previous results showing that MT can prevent Dox-induced cardiotoxicity.

Currently, most evidence suggests the generation of ROS as the culprit of Dox-induced cardiotoxicity. These ROS can be transformed into the more potent hydroxyl radical HO·, which can induce DNA and protein damage, membrane lipid peroxidation, and eventually lead to cell death and cardiac damage. ^28,29 Increasing evidence suggests that MT can function as a potent antioxidant in protection against oxidative stress in the cardiovascular system. ^{30 –33} Levels of ROS production were examined in the present study as a marker of oxidative stress. The results showed that intracellular ROS was significantly increased after Dox treatment, especially in MT^−/− cardiomyocytes. Our results also showed that the level of lipid peroxidation and protein carbonylation was markedly elevated by Dox in MT^−/− cardiomyocytes compared with MT^+/+ cardiomyocytes. These findings clearly indicate that MT provides protection against Dox-induced cardiotoxicity, which is possibly associated with inhibition of ROS generation.

The JAK-STAT pathway is a newly discovered intracellular signal-transducing pathway and plays a critical role in transduction of signal from the cell surface directly to the nucleus. ^12,34 Except as a regulator of cytokine signaling, now the JAK-STAT pathway has been confirmed to be an important membrane-to-nucleus signaling pathway for a variety of stress responses including oxidative stress, anticancer, cardiac hypertrophy, and ischemia–reperfusion. Recent studies have also shown that the JAK-STAT pathway performs some protective functions in cardiac hypertrophy and ischemia–reperfusion. ^35,36 A study demonstrated that STAT3-mediated signaling is proposed as a therapeutic target of ROS-induced cardiomyocyte injury. ³⁷ Another study demonstrated that the overexpression of STAT3 in the heart provides protection against Dox-induced cardiomyopathy. ¹⁸ It was also reported that the promoter region of the MT gene contains functional STAT3-regulatory sequence, providing a causality between STAT3 activation and MT induction. ¹⁹ In the present study, Western blot analysis indicated that Jak2 and Stat3 were phosphorylated after Dox treatment in MT^+/+ cardiomyocytes. We also observed that MT-1 and MT-2 mRNA and its protein expressions were both significantly upregulated after Dox treatment, however, AG490 can obviously prevent this upregulated situation in MT^+/+ cardiomyocytes. These findings strongly suggest that the JAK2/STAT3 pathway is initially activated through ROS release and then it mediates the upregulation of MT expression. The current study also demonstrates that AG490, the JAK2 inhibitor, can suppress the MT protection against Dox-induced toxicity in MT^+/+ cardiomyocytes, although this suppression is not apparent in MT^−/− cardiomyocytes. These results are consistent with a previous study that demonstrated the specific activation of STAT3 can transduce antioxidative signals through MT and confer resistance against ROS stress in the heart. ³⁴ These findings strongly support that the JAK-STAT pathway, especially the JAK2/STAT3-dependent pathway, can play a critical role in the protection of MT against Dox-induced cytotoxicity.

In summary, the present study strongly demonstrates that the JAK2/STAT3 pathway, at least in part, participates in MT protection against Dox-induced cardiotoxicity. Thus, it provides a possible mechanism for MT against Dox-induced cardiotoxicity. Such studies may contribute to the improvement in treatments for patients with cancer who are sensitive to Dox-induced cardiotoxicity.