Comparative proteomics analysis reveals role of heat shock protein 60 in digoxin-induced toxicity in human endothelial cells

Biochimica et Biophysica Acta 1784 (2008) 1857–1864

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a p a p

Comparative proteomics analysis reveals role of heat shock protein 60 in digoxin-induced toxicity in human endothelial cells Jie Qiu a, Hai-Qing Gao a,⁎, Ying Liang b, Han Yu c, Rui-Hai Zhou a a b c

Department of Geriatrics, Qilu Hospital of Shandong University, 107 Wenhua Xi Road, Jinan 250012, PR China Department of Geriatrics, Shandong Qianfoshan Hospital, Jinan 250014, PR China Department of Medical Microbiology, School of Medicine, Shandong University, Jinan 250012, PR China

a r t i c l e

i n f o

Article history: Received 24 April 2008 Received in revised form 18 June 2008 Accepted 8 July 2008 Available online 18 July 2008 Keywords: Digoxin Proteomics Toxicity Heat shock protein 60 Apoptosis Human endothelial cells

a b s t r a c t Although digitalis has been used in clinical treatment extensively, the precise mechanism of its toxic actions on cardiovascular system remained unclear, it would be of interest to study the differential proteomic analysis of vascular endothelial cells in response to toxic concentrations of digitalis thus to provide new agents for treatment of digitalis-induced cytotoxicity. We employed human umbilical vein endothelial cells (HUVEC) as our model system. HUVEC were exposed to increasing concentrations (0.1 nM–10 μM) of digoxin at 12–96 h intervals. Cell viability tests revealed that digoxin played dual effects on cell growth. Apoptosis detection confirmed that apoptosis was primarily responsible for digoxin-induced cell death. Proteomics analysis further revealed that the digoxin-induced apoptosis was accompanied by regulated expression of ATP synthase beta chain, cystatin A, electron transfer flavoprotein, heterogeneous nuclear ribonucleoproteins H3, lamin A, profilin-1, proteasome subunit 5, succinyl-CoA ligase beta chain and heat shock protein 60 (HSP60). Deep study on the overexpression of HSP60 confirmed that HSP60 exerted a protective role in digoxin-induced apoptosis through inhibition of caspase-3 activity in HUVEC. These results provided an impetus for further delineation of mechanism of digoxin-induced cytotoxicity and offered new agents that help attenuate its toxicity. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Digitalis has been used to treat congestive heart failure for more than 200 years, both the efficacy and the safety of this class of drugs continue to be a topic of debate [1], it is evident that it is prone to induce cytotoxicity, and its use in clinical practice is greatly limited by its associated adverse effects. Digitalis has been suggested to exert its actions through its ability to bind to and inhibit plasma membrane Na+–K+ATPase, and has the result of the increase in cellular [Ca2+] responsible for its positive inotropic action and its toxicity as well. Evidence was provided in several investigations that digitalis exerted dual effects on cell growth (proliferation and death) in different cell types, it has been observed to regulate several cellular functions, such as cell proliferation, apoptosis and antiapoptosis, in a variety of cells [2–5], the cytotoxic effect of digitalis has been observed on human lymphocytes, Jurkat cells, cortical neurons, human prostate cancer cells, and epithelial cells from the Madin–Darbin canine kidney [6–8]. However, despite these intensive studies, the precise biochemical mechanism by which digitalis induces cytotoxicity has not been fully defined; the form and mechanisms of cell death are not well defined ⁎ Corresponding author. Tel.: +86 531 866 77788; fax: +86 531 869 27544. E-mail address: [email protected] (H.-Q. Gao). 1570-9639/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2008.07.006

although the cytotoxicity induced by digitalis has been known for many years [9,10]. The cellular effects of exposure to digitalis are diverse, the cell damage may occur through more than one mechanism. All of these remained unclear. Two-dimensional gel electrophoresis (2-DE) is one of the most commonly used separation techniques in proteomics and is widely used in comparative studies of protein expression patterns in cells between different states. Recently, 2-DE based proteomic approaches have been utilized to identify new drug targets as well as drug toxicity [11,12]. As we have known, vascular endothelial cells, whose functional integrity is crucial for the maintenance of blood flow and the anti-toxicity activity, could be a target for digitalis; at the same time, one of the most dominant ways of the cytotoxicity of drugs could be related to the induction of programmed cell death of vascular endothelial cells; our previous study also had indicated that digitalis exerted both proliferation and apoptosis effects on human umbilical vein endothelial cells (HUVEC) [13]. Digoxin, the most commonly used preparation of digitalis, which is obtained from the leaves of Digitalis lanata (a common flowering plant called “foxglove”), plays a vital role in the therapy of congestive heart failure. Thus, we are interested in determining the mechanism of cytotoxicity effect of digoxin on HUVEC that may help identify potential subcellular targets involved in digoxin-triggered toxicity and signature events that can be

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exploited for therapies against such pathologies thus to offer new agents that help attenuate its toxicity in order to expand the application of digitalis The aim of this work was to characterize the toxicity effect of digoxin on cell growth in HUVEC, and, to do the differential proteomic analysis of HUVEC in response to toxic concentrations of digoxin and examine changes in protein expression. Deep study on the role of HSP60 indicated that HSP60 played a protective effect on HUVEC apoptosis induced by digoxin through inhibition of caspase-3 activity, so as to offer new agents that can help attenuate its toxicity in order to expand the application of digitalis. 2. Materials and methods 2.1. Materials Digoxin, DMSO and MTT were purchased from Sigma. Ac-DMQDCHO, a specific caspase-3 inhibitor, was purchased from Calbiochem, Darmstadt, Germany. The lactate dehydrogenase (LDH) kit and AnnexinV-FITC Apoptosis detection kit were obtained from BD Co. Ltd. EnzChek Caspase-3 assay kit was purchase from Molecular Probes Invitrogen. Human umbilical vein endothelial cells (HUVEC) were obtained from American Type Culture Collection (U.S.A.). Cell culture medium was from Gibco (U.S.A.). 2-DE reagents were obtained from Amersham Biosciences unless otherwise indicated. Mouse monoclonal IgG antibody to HSP60 was provided by R&D Systems, secondary antibody goat antimouse IgG was purchased from Santa Cruz Biotechnology. All other chemicals were of the highest purity and were available from commercial sources. Digoxin was dissolved in DMSO and diluted so that the final concentration of DMSO was b0.1%. All the control cells were treated with same volume of DMSO. 2.2. Cell culture and transduction of adenoviral constructs Primary cultures of HUVEC were according to a protocol we previously described [13]. Digoxin was dissolved in DMSO and diluted so that the final concentration of DMSO was b0.1%, all the control cells were treated with same volume of DMSO, which caused no effect on cell growth. The construction of recombinant adenoviruses expressing HSP60 was previously described [14,15]. In brief, the human HSP60 genes were cloned into the multiple cloning site of the adenoviral shuttle plasmid pACCMVpLpASR. Additionally, a recombinant adenoviral construct containing no insert (Adv−) was generated in parallel and used as a control. Viral constructs were plaque purified and propagated in human embryonic kidney 293 cells, and then purified by ultracentrifugation; the viral titers were determined by plaque assay

[16]. HUVEC were plated in 75 cm2 Petri dish in M199 containing 10% FBS and 1% penicillin/streptomycin. The cells were infected with adenoviruses of Ad-HSP60 for 48 h and incubated for indicated time in a 37 °C, 5% CO2 incubator. 2.3. Assay for MTT reduction and LDH release Cell viability was determined by MTT assay. MTT was added at a final concentration of 0.5 mg/ml, after being incubated at 37 °C for an additional 4 h, cells were centrifuged at 1000 rpm for 10 min and all the supernatants were discarded, the cells were dissolved in 120 μl isobutanol (containing 0.04 M HCl) and the absorbance was read at 570 nm, results were presented as the average absorbance and expressed as the mean of four samples. To measure the LDH release, HUVEC were incubated with either medium or medium containing the indicated concentrations of digoxin and were performed in the absence or presence of HSP60 for up to 96 h. Cell viability at the conclusion of these incubations was estimated by determining the release of LDH from cells after various incubations and at various times. LDH enzyme activity was measured using the LDH-cytotoxicity assay kit, according to the manufacturer's protocol. Test media were assayed in quadruplicate for each treatment condition. The total LDH activity was assessed by expressing as percentage LDH leakage (LDH in medium/total LDH activity × 100), respectively. 2.4. Analysis of apoptosis by flow cytometry To verify the form of HUVEC cell death induced by digoxin, cell cycle analysis and determination of cell apoptosis were performed by using AnnexinV-FITC Apoptosis Detection Kit. HUVEC were washed in Dulbecco's PBS and resuspended in binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 0.25 mM CaCl2) with annexin V-FITC and 2.5 μg/ml propidium iodide. After incubation at room temperature for 15 min in the dark, the fluorescence emitted by cells was analyzed with a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA), and viable cells (annexin-V negative, propidium iodide negative), cells in early apoptosis (annexin-V positive, propidium iodide negative), and cells in late apoptosis or necrosis (annexin-V positive, propidium iodide positive) were identified and counted, respectively. A minimum of 5000 cells were counted for each sample. 2.5. Determination of caspase-3 activity To detect apoptotic events, caspase-3 enzyme activity was determined in HUVEC. The treated cells were washed twice with PBS and harvested in 200 μl lysis buffer (100 mM NaCl, 10 mM Tris, pH 7.5,

Fig. 1. Effect of digoxin on cell viability. (A) Effect of digoxin on HUVEC cell growth. Cells (3 × 103 in 100 μl) cultured in 96-well plates were exposed for 12 h, 24 h, 48 h and 96 h at indicated concentrations of digoxin. Control group: medium plus DMSO at the same concentration employed in treated cultures was added. Final concentrations of digoxin are shown on the X-axis. Cell number was determined as described under Materials and methods. Results are expressed as relative cell number (%) with reference to control. Data are expressed as mean ± SEM values from four independent experiments. ⁎p b 0.01 compared to control cells. (B) Picture of transmission electron microscope showing the morphology of HUVEC in control or digoxin-treated cells. (a) Control cells, (b) Apoptosis cells in the treatment with 210 nM digoxin for 48 h, injured cells show apoptotic features such as highly condensed nuclei and apoptotic bodies (arrows). M, mitochondria; N, nucleus; rER, rough endoplasmic reticula.

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1 mM EDTA, and 0.01% Triton X-100). The cell lysate was centrifuged at 12,000 g for 20 min and the clear supernatant was used for the analysis. Caspase-3 activity was measured using the EnzChek Caspase3 assay kit (Molecular Probes), according to the manufacturer's instructions. Fluorescence was measured at 485 nm for excitation and 535 nm for emission with a multilabel plate counter. Caspase-3 activity was expressed as arbitrary units of fluorescence signal normalized for the protein content. 2.6. Two-dimensional gel electrophoresis (2-DE), protein detection and image analysis

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transferred to polyvinylidene difluoride (PVDF) membranes. The membrane was blocked by incubation in 5% non-fat milk in trisbuffered saline and incubated with primary antibodies overnight at 4 °C. Blots were developed using peroxidase-conjugated secondary antibodies and visualized with enhanced chemiluminescence reagent (Amersham Pharmacia Biotech, Buckinghamshire, UK) according to the manufacturer's recommendations. Bands were quantified with Imagequant software, using western blotting of β-actin (clone AC-15, Sigma) as a loading control; blots were incubated with anti-HSP60 (MAB1800, R&D Systems). 2.9. Statistics analysis

The sample protein levels were assessed using the protein assay PlusOne 2-D Quant kit with BSA as the standard. 2-DE was performed using 18 cm, non-linear, pH 3–10, immobilized pH gradient (IPG) strips (GE Healthcare) and 12% bonded gels exactly as described by our previous work [13]. After the second dimension, gels were stained with silver according to Yan had reported [17]. Briefly, the gels were fixed in 50% ethanol and 5% acetic acid and then sensitized in 0.02% sodium thiosulfate. The staining was performed in 0.1% silver nitrate. Dried 2-D gels were scanned with a HP Scanjet 7400c (Hewlett Packard), protein spots were quantified and numbered using the PDQuest Image Analysis software (Bio-Rad) and checked manually to eliminate artifacts due to gel distortion, abnormal silver staining or poorly detectable spots. After background subtraction, normalization, and matching, the spot volumes in gels from each control cells were compared with the matched spot volumes in gels from digoxin-treated cells. The protein level of each spot was expressed as a percentage of total spot volume in the 2-DE gel. Comparison of test spot volumes with corresponding standard spot volumes gave a standardized abundance for each matched spot and values were averaged across triplicates for each experimental condition. Statistical analysis was performed to pick spots matching across all images, spots displaying a ≥ 1.5 average-fold increase or decrease in abundance between conditions, matching across all gel images and having P valuesb0.05.

Data are expressed as means ± standard error of the mean (S.E.M.) based on data derived from three to six independent experiments. The statistical significance was tested by paired Student's t-test or by

2.7. Mass spectrometry Single protein spots from the digoxin-treated HUVEC were excised from a representative gel stained with silver and submitted for tryptic digestion and LC–MS/MS to Proteomics International. Protein spots were in-gel digested by manually as Shevchenko has reported [18]. The tryptic digests were assessed by liquid chromatography electrospray ionization mass spectrometry/mass spectrometry (LTQ– ESI–MS/MS, ThermoFinnigan, San Jose, CA), and performed using a Surveyor high-performance liquid chromatography (HPLC) system connected through PepFinder kit (with peptide trap and 99:1 flow splitter), the ion transfer capillary temperature was set at 170 °C. Mass data collected during the LTQ–ESI–MS/MS analysis were processed and converted into a file using the Masslynx™ software (Micromass) to be submitted to the Mascot search software (http://www. matrixscience.com/). Protein identification was obtained by comparison of experimental data with the NCBI non-redundant mammalian database and was validated when considering at least two peptide sequences per protein. The main cellular location and function of identified proteins, as well as their respective biological relationships, were searched and determined by going through PubMed http://www.ncbi.nlm.nih.gov/ entrez/query.fcgi) with EndNote software v.5.0. 2.8. Western blot analysis Equal amounts of conditioned-medium proteins (20 μg) were loaded into a 12% polyacrylamide gel and electrophoresed as described previously. In brief, proteins were separated by SDS-PAGE and

Fig. 2. Digoxin induces toxicity in HUVEC. HUVEC were incubated with the indicated concentrations of digoxin for the indicated times. Effect of digoxin on the cytotoxicity of HUVEC measured by MTT assay (A, B) and LDH release (C).The effects of MTT reduction and LDH release were measured as described under Materials and methods for the cytotoxicity, respectively. Concentration-dependent (A) and time-dependent (B) effects to digoxin action in HUVEC, the viability of HUVEC were detected by MTT assay. (A) Cells were exposed to increasing concentrations of digoxin for 48 h. (B) Cells were treated with digoxin 210 nM for various periods. (C) Concentration-dependent and time-dependent LDH release induced by digoxin in HUVEC. Results are mean ± SEM of measurements from four different experiments. ⁎p b 0.01 compared to control group.

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trypan blue dye exclusion and cell survival was estimated with MTT assay. An increase in cell population was observed when HUVEC were exposed to digoxin for 24 h at concentrations ≤ 10 nM compared with the control cells. The proliferative effect of nontoxic concentrations (≤20 nM) of digoxin on HUVEC growth was time- and concentrationdependent (data not shown); the digoxin cytotoxic effect was evident when cells were incubated with high concentrations of digoxin. Incubation of HUVEC with digoxin at concentrations ranging from 0.1 μM to 10 μM for 24 h, 48 h and 96 h resulted in a dose-related reduction in cell viability of HUVEC cultured in M199 supplemented with 10% fetal bovine serum as shown in Fig. 1A, at 48 h of exposure, digoxin decreased cell populations significantly at 210 nM or higher concentrations, the inhibitory action of digoxin on HUVEC growth was more pronounced when exposure lapse was increased to 96 h. 3.2. Digoxin induces cytotoxicity at high concentrations and late incubation times

3. Results

We investigated the sensitivity of HUVEC to digoxin induced cytotoxicity by incubating cells with digoxin from 40 nM up to 10 μM for up to 96 h. We explored the concentration–response relationships for cytotoxicity produced by digoxin. Incubation of confluent HUVEC with digoxin clearly induced cytotoxicity as shown by the decrease of MTT activity, a marker of cell death and loss. 320 nM digoxin for 48 h caused a 65% loss of MTT activity, while 210 nM digoxin for 48 h resulted in a 55% loss of MTT activity (Fig. 2). Microscopic observation confirmed the extensive cell loss and agreed with the reduction in MTT activity (as shown in Figs. 1B and 2A, B). Then we determined the LDH release induced by digoxin, data showed that digoxin-induced LDH release was in a time-dependent and dose-dependent manner, cells exposed to 320 nM digoxin for at least 24 h were required to produce at least 50% LDH release and 40 nM for 96 h increased LDH release markedly increased LDH release to 71.5% as shown in Fig. 2C. Hence, digoxin-induced toxicity in primary cultures of HUVEC was at a relatively high-dose and long-time response.

3.1. Effect of digoxin on HUVEC viability

3.3. Effect of digoxin-induced apoptosis in HUVEC

In order to assess the effect of digoxin on cell growth in HUVEC, the viable cell number was measured by microscopic examination of

It has been demonstrated that digoxin is capable of inducing apoptosis death in Hela cells and prostate cancer cells [19,20], and in

Fig. 3. Cell apoptosis and caspase-3 activity were analyzed in digoxin-treated cells. (A) HUVEC cells were incubated with control condition (a) or exposed to 210 nM digoxin for 48 h (b). The cell apoptosis was assessed by flow cytometer with annexin V-FITC and propidium iodide (PI) and represented in the scatter plot (a, b). Vertical and horizontal lines in the graph are designed based on autofluorescence of untreated control cells. Figures in lower right quadrant indicate percentage of apoptotic cells. (B) Caspase-3 activity was detected as described in material and methods part. Mean ± SEM obtained from experiments performed in quadruplicate are given. ⁎p b 0.01 compared to control cells, ⁎⁎p b 0.01 digoxin-treated cells vs. inhibitor pre-treated cells.

one-way ANOVA followed by Fisher's test when appropriate. A P value below 0.05 was considered statistically significant.

Fig. 4. Protein 2-DE maps of digoxin-treated HUVEC and control cells. Gels were stained by silver. (A) 2-DE profiles of total proteins extracted from the control cells. Comparison of both protein profiles revealed that there were many protein spots regulated. Nearly 3000 protein spots were visualized, 9 proteins were identified by LTQ–ESI–MS/MS. The number in gels is the spot identified. (B) Picture of HUVEC with 210 nM digoxin-treated at 48 h, the dots marked the variation proteins.

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times, and digoxin enhanced caspase-3 activity at high concentrations with longer time exposures. To confirm that digoxin-mediated activation of caspase-3 is responsible for the observed apoptosis, a specific caspase-3 inhibitor Ac-DMQD-CHO (100 μM) was added to cell cultures 90 min before digoxin treatment. The results revealed that 48 h of 210 nM digoxin treatment increased caspase-3 activity by approximately 2.8-fold as compared with the control and this enhancement was decreased by pretreatment with Ac-DMQD-CHO (Fig. 3B). This indicated that apoptosis induced by digoxin was at least partially mediated by caspase-3 activation in HUVEC. 3.4. Changes in protein profile of HUVEC following digoxin-induced cell death

Fig. 5. Change in protein expressions in HUVEC exposed to digoxin at 210 nM for 48 h. (A) Close-up images of several regions of the 2-DE gels showing representative differentially expressed protein spots. Each panel (a–i) is representative pairs of images from the control cells (left) and the digoxin-treated cells (right) (a) Lamin A, (b) HSP60, (c) ATP synthase beta chain, (d) Succinyl-CoA ligase beta chain, (e) Heterogeneous nuclear ribonucleoproteins H3, (f) Proteasome subunit 5, (g) Cystatin A, (h) Profilin-1, (i) Electron transfer flavoprotein. (B) The differentially expressed proteins between digoxin-treated HUVEC and control cells as determined by 2-DE and LTQ–ESI–MS/MS. The up-regulated proteins in HUVEC are showed above the X-axis and the downregulated proteins are showed below the X-axis. Number in the parenthesis corresponds to that in panel A of this figure.

the present work, the addition of digoxin to cultured HUVEC in the concentrations above 0.1 μM resulted in an increased amount of apoptotic cells. As shown in Fig. 3, the percentage of apoptosis was analyzed quantitatively by flow cytometry. A significant increase in the apoptotic rate (from 4.4% to 52.7%) was observed compared to control group after the cells were treated with 210 nM digoxin for 48 h (Fig. 3A). Thus, we confirmed that apoptosis was primarily responsible for the observed HUVEC cell death triggered by digoxin. The activation of proteases such as caspase-3 is an important effect in cellular apoptosis, caspase-3 activity was detected in samples treated with different concentrations of digoxin for different exposure

The purpose of this work was to identify in HUVEC the changes in protein expression induced by the toxic action of digoxin. A total of nearly 3000 protein spots were mapped in pairs on control group and digoxin-treated HUVEC gels and the identity of protein spots were identified by tryptic digestion followed by LTQ–MS–MS analysis as shown in Fig. 4. Detailed gel analysis revealed 9 reproducible protein spots with more than 2-fold change in density between the two gels, 6 proteins were up-regulated and 3 proteins were downregulated in digoxin-treated HUVEC. As showed in Fig. 5, the expression of ATP synthase beta chain (spot 3) , cystatin A (spot 7), heterogeneous nuclear ribonucleoproteins H3 (spot 5), lamin A (spot 1), proteasome subunit 5 (spot 6) and succinyl-CoA ligase beta chain (spot 4) were significantly increased, whereas that expression of protein electron transfer flavoprotein (spot 9) , HSP60 (spot 2) and profilin-1 (spot 8) were significantly down-regulated in digoxintreated cells, the identified proteins involved in various aspect of endothelial cellular function, including metabolism, cell motility and signal transduction as well (as shown in Table 1), most of these proteins have already been described as modulators of various apoptotic processes. 3.5. Effect of digoxin on the expression of HSP60 To further confirm the results of 2-DE, we carried out western blot analysis of HSP60, and digoxin decreased the expression of HSP60 in HUVEC (Fig. 6A), met the result of 2-DE as showed in Fig. 5. 3.6. HSP60 attenuates toxicity induced by digoxin in HUVEC To determine whether HSP60 can suppress apoptosis induced by digoxin, HSP60 was overexpressed in the HUVEC transduced with the adenovirus carrying HSP60, whereas the expression of HSP60 was not altered in the cells transduced with the control virus (Fig. 6). In order to investigate whether HSP60 may be involved in the cytotoxicity induced by digoxin in HUVEC, cell viability was measured after cells were infected with Ad-HSP60. When HUVEC cells were infected with Ad-HSP60 before exposed to 210 nM digoxin for 48 h,

Table 1 Identify of the nine proteins regulated in association with digoxin-induced apoptosis in HUVEC Protein ID

Numa

MW (Da)b

pIb

NCBI accession

Location

Function

ATP synthase beta chain Cystatin A Electron transfer flavoprotein HSP60 Heterogeneous nuclear ribonucleoproteins H3 Lamin A Profilin-1 Proteasome subunit 5 Succinyl-CoA ligase beta chain

3 7 9 2 5 1 8 6 4

56559.96 11066.44 35079.53 61212.59 35238.68 74139.71 14922.95 17328.08 46510.56

5.26 5.38 8.62 5.7 6.36 6.57 8.47 5.94 6.15

P06576 P01040 P13804 P10809 P31942 P02545 P07737 Q99471 Q96I99

Mitochondrion Cytoplasm Mitochondrion Mitochondrion Nucleus Nucleus Cytoplasm Cytoplasm; nucleus Mitochondrion

ATP production Cellular metabolism Electron transfer: respiration Stress response Heat shock stress response Nuclear envelope component Cytoskeleton: cell mobility Cellular metabolism ATP production

a b

Number of the protein spot in the 2-DE master gel. Predicted MW and pI from sequence analysis.

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cell death rates decreased to 1.0 and 2.0-fold compared to digoxintreated cells, and, the cells were infected with adenoviruses of Advshowed no significant differences, thereby providing further evidence that overexpression of HSP60 attenuates cytotoxicity triggered by digoxin in HUVEC. The results indicated that HSP60 exerted its protective role in digoxin-triggered toxicity. To further confirm the effect of HSP60 on toxicity induced by digoxin in HUVEC, MTT reduction and LDH release were measured after HSP60 were overexpressed in HUVEC, the results showed that overexpression of HSP60 inhibited the digoxin-induced LDH release significantly (Fig. 6C) and partially recovered the MTT reduction (Fig. 6B), it was interesting that the protective effect was greater in HUVEC cytotoxity triggered by digoxin with long time than with short time exposures. 3.7. Protective effect of HSP60 on digoxin-induced apoptosis in HUVEC To evaluate the protective effect of HSP60 on digoxin-induced cell apoptosis, the presence of apoptotic cells infected with adenoviruses of Adv-(control cells), or Ad-HSP60 for 48 h was further confirmed by flow cytometry. As shown in Fig. 7A, HSP60 attenuated the digoxininduced apoptosis in HUVEC cells. On the other hand, a similar result was obtained when HUVEC were pre-treated with a caspase-3 inhibitor Ac-DMQD-CHO. HSP60 and Ac-DMQD-CHO caused 60.7% and 64.3% inhibition of apoptosis in digoxin-treated HUVEC cells, respectively, there was on significant difference between overexpression of HSP60 and caspase-3 inhibitor (Fig. 7B). The results thus demonstrated that HSP60 attenuated digoxin-triggered cytotoxicity induced by decreasing cell apoptosis.

Fig. 7. Overexpression of HSP60 mediates digoxin-induced apoptosis through inhibition of caspase-3 in HUVEC. (A) The cell apoptosis was assessed by flow cytometer with annexin V-FITC and propidium iodide (PI) and represented in the scatter plot (a–e). HUVEC were infected with Adv-incubated in medium alone served as control (a), HUVEC were incubated with digoxin (210 nM for 48 h) (b), cells were infected with AdHSP60 (c), cells were preincubated with inhibitor (d) and cells were infected with AdHSP60 and preincubated with inhibitor (e) as analyzed by dual color flow cytometry using annexin V-FITC and PI. (B) Data show the mean ± SEM (a–e). (⁎p b 0.01, #p N 0.05). (C) HUVEC cells were infected with Ad-HSP60 for 48 h or incubated with caspase-3 inhibitor for 90 min before incubated with digoxin or without digoxin, at various concentrations for 48 h. Mean ± SEM obtained from experiments performed in quadruplicate are given. ⁎p b 0.01 cells infected with Add-HSP60 before treated with digoxin compared to digoxin-treated c3ells.

3.8. Effect of overexpression of HSP60 on digoxin-induced activation of caspase-3

Fig. 6. Digoxin-induced toxicity is attenuated by overexpression of HSP60 in HUVEC. (A) Western blot analysis of HSP60 expression in HUVEC. HUVEC were treated with 210 nM digoxin, each aliquot of the cell extracts and culture media were taken at the indicated times and analyzed for MTT reduction (B) and LDH release (C), respectively. ⁎p b 0.01, ⁎⁎p b 0.001. Methods were described under Materials and methods, respectively. Results are mean ± SEM of measurements from four different experiments.

To define the effect of HSP60 overexpression on digoxin-induced apoptosis through caspase-3 pathway, we have investigated activation of caspase-3 in the cells transduced with control viral vector and AdHSP60. The results revealed that 48 h digoxin treatment increased caspase-3 activity in a concentration-dependent manner and overexpression of HSP60 dramatically inhibited activation of caspase-3 (Fig. 7C). These findings showed that overexpression of HSP60 in

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HUVEC directly mediated digoxin-induced apoptosis through caspase3 pathway. 4. Discussion Digitalis has played a prominent role in the therapy of congestive heart failure since William Withering codified its use in his classic monograph on the efficacy of the leaves of the common foxglove plant in 1785[21]. However, data relating to its toxicity are scarce. Digoxin is by far the most commonly prescribed of these drugs, several studies have demonstrated that it had dual effects on cell growth such as vascular smooth muscle cells, lymphocytes, prostate cells, and Hela cells [19,20,22,23]. In this work, we demonstrated that digoxin played a bifunctional role in HUVEC growth, the cytotoxicity produced by digoxin was analyzed by MTT assay and LDH release, and the results showed that the toxic effect of digoxin was time- and concentration-dependent, cells affected by toxic dosages of digoxin shrank and presented nuclear condensation and apoptotic bodies as shown by transmission electron microscope (Fig. 1B), all characteristic changes of apoptotic cell death. Flow cytometry confirmed that apoptosis was primarily responsible for the cell death induced by digoxin, meanwhile, detection of caspase-3 activation indicated that in the cell death induction mechanism existed mitochondrial damage with consequent caspase-3 activation, similar to the results of Ramirez-Ortega's study on the apoptosis triggered by digoxin in Hela cells [20]. These results also supported our opinion that the proteins which identified by proteomics approach may play critical role in digoxin-triggered apoptosis. To further investigate the biochemical mechanism of apoptosis induced by digoxin, we tried to detect the altered proteins involved in HUVEC apoptosis triggered by digoxin by using proteomics approaches. As shown in Table 1, the functional characterization of the 9 identified proteins confirmed important cellular functions such as metabolism, cell contractility and resistance to chemical or physical “stress” (Table 1 and Fig. 5). Overall, the regulation of proteins involved in ATP production such as ATP synthase beta chain, electron transfer flavoprotein and succinyl-CoA ligase beta chain [24] might be correlated with the need for ATP to produce more stress response heterogeneous nuclear ribonucleoproteins H3 protein and cellular metabolism protein-Cystatin A [25,26]. At the same time, over expression of proteasome subunit 5 increased the amount of proteasome and also ameliorated cell response to stress [27]; this may be correlated to the down-regulated HSP60. On the other hand, the digoxin-induced apoptosis also included cytoskeleton rearrangement, Ding just reported the silencing profilin-1 inhibits endothelial cell proliferation [28]; the result was accordance with our study. At the same time, the up-regulated protein lamin A [29], one of nuclear membrane proteins, played important role in digoxintriggered apoptosis by means of nuclear dysfunction. 2-D gel electrophoresis combined with mass spectrometry in this respect is a useful first approach to get an overview of proteomic alterations in digoxin-treated HUVEC. Findings of interesting proteins then will be verified and further study on the function of identified proteins is under way. Based on the gel separation results, we focused our studies on HSP60. Heat shock proteins play an important role in helping cells cope with a number of stresses such as heat shock and oxidative stress [30]. The increased production of heat shock proteins is a central and protective cellular response to environmental and metabolic stress in all procaryotic and eucaryotic organisms. Importantly, the HSP60 has been shown to have the ability to regulate apoptosis by interacting, directly or indirectly, with a number of apoptotic proteins [15]. As discussed above, our results showed that the changed expression of HSP60 was correlated with other proteins involved in signaling pathways of cell growth. HSP60 is generally considered to act in an anti-apoptotic manner due to its role in the inhibition of the activa-

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tion of caspases; however, some researchers reported that HSP60 was pro-apoptotic by promoting activation of procaspase-3 but also anti-apoptotic by preventing Bax and Bak translocation to the mitochondria [31,32,33]. Thus, the role of HSP60 in apoptosis may be both pro- and anti-apoptotic, possibly depending on the cell type, and provoking stimulus for apoptosis. In our experiments, we confirmed that HSP60 was down-regulated when HUVEC were incubated with toxic concentrations of digoxin by using proteomics approach, the result was similar to the study on the protective effect of over expression of HSP60 on cardiac cells and HaCaT cells, while different from what reported in Jurkat cells [31], sufficient reasons exist for thinking that the initial step is the differential effects of HSP60 on apoptosis in different cell lines and inductive apoptosis conditions. Among our hypotheses was the idea that over expression of HSP60 would protect the cell against apoptosis induced by digoxin. Our data indicated that cell death associated with apoptosis was decreased with overexpression of HSP60 (Fig. 6); furthermore, the results of flow cytometry and detection of caspase-3 activity confirmed the protective effect of HSP60 on apoptosis was accordant with inhibition of caspase-3 activity (Fig. 7). Our results were somewhat similar to those of Zhang et al. [34], who observed that bacterial HSP60 suppressed apoptotic cell death via inhibiting caspase-3 activity, while our data further supported that HSP60 played a protective role in preventing digoxin-triggered cytotoxicity by using proteomics approach. In summary, to our knowledge this is the first report demonstrating the role of HSP60 in digoxin-induced apoptosis in HUVEC by proteomics approach. Previous and current results thus indicate that the differently expressed proteins may be useful as an independent marker for digoxin toxicity; our study supports the concept that proteins may be used as pharmacological agents to control cell apoptosis during digitalis triggered apoptosis and amend adverse effects. Further confirmation and validation of the candidate proteins in HUVEC apoptosis induced by digoxin will provide an opportunity to assign novel toxic markers and development of therapeutic strategies. Acknowledgements This work was supported by research grants from the National Natural Science Foundation of China (No: 30700884) and Foundation of Science and Technology Development of Shandong Province, P.R.C (No: Q2005C01 and No: 2007GG10002018). The authors gratefully acknowledge the technical assistance of the personnel in “Research Centre for Proteome Analysis, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences”. References [1] J.A. Wasserstrom, G.L. Aistrup, Digitalis: new actions for an old drug, Am. J. Physiol.: Heart. Circ. Physiol. 289 (2005) H1781–1793. [2] J. Qiu, H.Q. Gao, B.Y. Li, L. Shen, Proteomics investigation of protein expression changes in ouabain induced apoptosis in human umbilical vein endothelial cells, J. Cell. Biochem. 104 (2008) 1054–1064. [3] S.N. Orlov, N. Thorin-Trescases, S.V. Kotelevtsev, J. Tremblay, P. Hamet, Inversion of the intracellular Na+/K+ ratio blocks apoptosis in vascular smooth muscle at a site upstream of caspase-3, J. Biol. Chem. 274 (1999) 16545–16552. [4] F.A. Verheye-Dua, L. Bohm, Influence of apoptosis on the enhancement of radiotoxicity by ouabain, Strahlenther. Onkol. 176 (2000) 186–191. [5] N.K. Isaev, E.V. Stelmashook, A. Halle, C. Harms, M. Lautenschlager, M. Weih, U. Dirnagl, I.V. Victorov, D.B. Zorov, Inhibition of Na(+),K(+)-ATPase activity in cultured rat cerebellar granule cells prevents the onset of apoptosis induced by low potassium, Neurosci. Lett. 283 (2000) 41–44. [6] S.P. Yu, Na (+), K (+)-ATPase: the new face of an old player in pathogenesis and apoptotic/hybrid cell death, Biochem. Pharmacol. 66 (2003) 1601–1609. [7] D. Pchejetski, S. Taurin, S. Der Sarkissian, O.D. Lopina, A.V. Pshezhetsky, J. Tremblay, D. deBlois, P. Hamet, S.N. Orlov, Inhibition of Na+,K+-ATPase by ouabain triggers epithelial cell death independently of inversion of the [Na+]i/[K+]i ratio, Biochem. Biophys. Res. Commun. 301 (2003) 735–744. [8] Y.T. Huang, S.C. Chueh, C.M. Teng, J.H. Guh, Investigation of ouabain-induced anticancer effect in human androgen-independent prostate cancer PC-3 cells, Biochem. Pharmacol. 67 (2004) 727–733.

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