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Am J Physiol Heart Circ Physiol 295: H907-H913, 2008. First published June 13, 2008; doi:10.1152/ajpheart.00279.2008
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Factors released from embryonic stem cells inhibit apoptosis in H9c2 cells through PI3K/Akt but not ERK pathway

Dinender K. Singla,1 Reetu D. Singla,1 and Debbie E. McDonald2

1Biomolecular Science Center, Burnett School of Biomedical Sciences, College of Medicine, Orlando, Florida; and 2Department of Medicine, University of Vermont, College of Medicine, Burlington, Vermont

Submitted 14 March 2008 ; accepted in final form 9 June 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We recently reported that embryonic stem cells-conditioned medium (ES-CM) contains antiapoptotic factors that inhibit apoptosis in the cardiac myoblast H9c2 cells. However, the mechanisms of inhibited apoptosis remain elusive. In this report, we provide evidence for the novel mechanisms involved in the inhibition of apoptosis provided by ES-CM. ES-CM from mouse ES cells was generated. Apoptosis was induced after exposure with H2O2 (400 µm) in H9c2 cells followed by the replacement with ES-CM or culture medium. H9c2 cells treated with H2O2 were exposed to ES-CM, and ES-CM plus cell survival protein phosphatidylinositol 3-kinase/Akt inhibitor, LY-294002, or extracellular signal-regulated kinase (ERK1/2) inhibitor, PD-98050. After 24 h, H9c2 cells treated with ES-CM demonstrated a significant increase in cell survival. ES-CM significantly inhibited (P < 0.05) apoptosis determined by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining, apoptotic ELISA, and caspase-3 activity. Importantly, enhanced cell survival and inhibited apoptosis with ES-CM was abolished with LY-294002. In contrast, PD-98050 shows no effect on ES-CM-increased cell survival. Furthermore, H2O2-induced apoptosis is associated with decreased levels of phosphorylated (p)Akt activity. Following treatment with ES-CM, we observed a decrease in apoptosis with an increase in pAkt, and the increased activity was attenuated with the Akt inhibitor, suggesting that the Akt pathway is involved in the decreased apoptosis and cell survival provided by ES-CM. In contrast, we observed no change in ES-CM-decreased apoptosis or pERK with PD-98050. In conclusion, we suggest that ES-CM inhibited apoptosis and is mediated by Akt but not the ERK pathway.

hydrogen peroxide; phosphatidylinositol 3-kinase; extracellular signal-regulated kinase

APOPTOSIS IS CONSIDERED to play a major role in the development and progression of myocardial infarction that leads to cardiac myocyte cell loss and then to heart failure (2, 3, 5, 21, 24). Cardiac myocyte cell loss in the infarcted heart is inhibited by antioxidants, angiotensin II inhibitors, and the expression of the antiapoptotic protein Bcl-2 (21, 24, 29). Both adult and embryonic stem (ES) cell transplantation studies demonstrated a decrease in cardiac myocyte apoptosis, limited cardiac regeneration with significant improved cardiac function (14–16, 20, 27, 28, 40, 41). Importantly, these studies suggest that autocrine or paracrine factors released in the heart following ES or adult stem cell transplantation inhibit host myocardium apoptosis (14, 15, 41). Using cell culture model, our laboratory (42) recently published direct evidence that factors released from ES cells contain antiapoptotic factors that inhibit H2O2-induced apoptosis in the cardiac myoblast H9c2 cells.

The mechanisms of inhibited apoptosis are associated with the activation of the cell survival-signaling cascades phosphatidylinositol 3-kinase (PI3K)/Akt or ERK1/2 (12, 37–39). The use of antiapoptotic agents, such as bradykinin, cardiotrophin-1, insulin, insulin growth factor-1, and urocortin, which reduce ischemia-reperfusion injury in animals, is associated with the upregulation of the phosphorylated (p)PI3K/Akt and pERK1/2 pathways (17). However, the mechanisms of H9c2 cells-inhibited apoptosis using factors released from ES cells are completely unknown.

Therefore, we hypothesize that the factors released from ES cells-inhibited apoptosis are mediated through cell survival/antiapoptotic pathways PI3K/Akt and ERK. We present these data for the first time that the factors released from ES cells-inhibited apoptosis are mediated through PI3K/Akt but not the ERK-signaling pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Preparation of ES cell-conditioned medium. Mouse ES cells (line CGR8) were passaged and maintained at our laboratory (42–44) reported previously. In brief, mouse ES cells are maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) containing leukemia inhibitory factor (LIF), sodium pyruvate, β-mercaptoethanol, penicillin-streptomycin, glutamine, nonessential amino acids, and 15% ES cell-qualified fetal bovine serum (Invitrogen) (42–44). We obtained rat cardiomyocyte-derived cell line H9c2 (H9c2 cells) from the American Type Culture Collection (Manassas, VA). Cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, and 25 µg/ml gentamicin at 37°C in a humidified atmosphere of 5% CO2-95% O2 as we reported previously (42).

Mouse ES cells at 9,000 ES cells/cm2 were grown in 0.5% gelatinized petri dishes containing cell culture medium with LIF for 24 h. Cells were then replaced with fresh cell culture medium without LIF. After 48 h, the supernatants were collected and labeled as ES-conditioned medium (CM). ES-CM was filtered (0.22 µm cellulose syringe filter) and used to determine the effects on H2O2-induced apoptosis in H9c2 cells.

Apoptotic cell culture model. H9c2 cells were cultured (2,000 cells/well) for 24 h. Our laboratory (42) reported previously (42) and used an optimal dose of 400 µm of H2O2 (Sigma). H9c2 cells were exposed with H2O2 (400 µm) for 2 h followed by a replacement with fresh cell culture medium or ES-CM or ES-CM + LY-294002 (40 µM) or ES-CM + PD-98050 (25 µM) (Calbiochem). We performed an apoptotic ELISA study using different concentrations of LY-294002 (20–80 µM) and PD-98050 (12.5–25 µM) to determine the optimal concentrations. The LY-294002 (40 µM) and PD-98059 (25 µM) concentrations used in the present study are well within the range of LY-294002 and PD-98059 (5–50 µM) used by other investigators in their cell culture studies (9, 13, 34, 42). After 24 h, control wells without H2O2 treatment were replaced with fresh cell culture medium. Cells were cultured for an additional 24 h and then counted in each of the 24 wells based on their rod-shaped morphology. Rod-shaped cells were considered to be alive, whereas round cells were considered to be dead. The number of rod shape cells in duplicate wells was counted. The mean of the duplicated wells was taken and used to analyze the data. Cell viability was also examined using trypan blue staining.

Using our cell culture model system, apoptosis was examined by the terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) staining, the apoptotic ELISA kit, and caspase-3 activities. Phosphorylated PI3K/Akt and ERK activities were measured using Superarray ELISA kits.

Trypan blue staining. H9c2 cells (5,000 cells) were cultured in a 35-mm petri dish for 24 h. Cells were treated with and without H2O2 and then replaced with cell culture medium, ES-CM, ES-CM + LY-294002, ES-CM + PD-98059, LY-294002, or PD-98059 for an additional 24 h. Cells were washed with PBS. Cells were enzymatically dissociated using trypsin-EDTA (GIBCO), washed with PBS, and were incubated with 0.4% trypan blue stain (Sigma) for 5 min at room temperature. Cells were counted in a hemocytometer chamber. In parallel, cells were also stained in the petri dish using trypan blue staining without trypsinization. The experiments were repeated five times in duplicate. Cells stained blue (dead) and unstained (live) cells were counted. The percentages of viable cells were calculated as the total number of live cells divided by the total number of cells and multiplied by 100.

TUNEL staining. TUNEL staining was performed to determine the apoptotic positive nuclei in H9c2 cells following exposure with the H2O2 and the respective treatment groups. H9c2 cells were washed with PBS and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS). Following fixation, the cells were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate followed by proteinase K (25 µg/ml in 100 mM Tris·HCl). An apoptotic cell death detection kit (TMR red, Roche Applied Science) based on the TUNEL assay was used to detect apoptotic cells according to the manufacturer's instructions. Each experiment included negative controls by omitting the TUNEL enzyme TdT reaction mixture and incubating the cells with the label solution provided in the kit. Cells were mounted with Antifade Vectashield mounting medium containing 4',6-diaminio-2-phenylindole (DAPI; Vector) to stain the total nuclei and were examined with a fluorescence microscope (Zeiss Axiovert 200). To calculate the percent apoptotic nuclei, we counted the number of TUNEL-staining red fluorescence nuclei divided by the total DAPI-positive blue fluorescence nuclei and multiplied by 100.

Cell death detection ELISA. The cell death detection ELISA plus kit obtained from Roche Applied Science was applied to measure the apoptosis based on the principle of histone-bound DNA fragments in an ELISA format. Medium was used as reported by our laboratory (42) and others (32) to examine the apoptosis collected at 24 h from different groups.

Caspase-3 activity assay. One million H9c2 cells were cultured in 150-mm2 petri dishes for 2 to 3 days. Cells were treated with and without H2O2 and then replaced with cell culture medium, ES-CM, ES-CM + LY-294002 (40 µM), or ES-CM + PD-98059 (25 µM) for an additional 24 h. Caspase-3 activity was measured as we reported previously using a caspase-3 colorimetric activity assay kit from BioVision. Cells were dissociated enzymatically with trypsin-EDTA (Invitrogen) and then collected in the cell lysis buffer provided in the kit. The cell lysate was prepared by incubating the cells on ice for 30 min and then centrifuging. Supernatant was isolated and used for protein concentration and caspase-3 activity. The protein concentrations were measured in the supernatant using a Bio-Rad assay. Caspase-3 activity was performed as per manufacturer's instructions provided in the kit, colorimetric reaction was developed, and measured at 405 nm in a microtiter plate reader.

Phosphorylated Akt and ERK activity. H9c2 cells (7,500 cells/well) were cultured in 96-well plates for 24 h. Cells were treated with and without H2O2 and then replaced with cell culture medium, ES-CM, ES-CM + LY-294002 (40 µM), or ES-CM + PD-98059 (25 µM) for an additional 24 h. Phosphorylated and total Akt and ERK activities were examined using commercially available kits (Case kit Akt and ERK, Superarray Biosciences, Frederick, MD). In brief, each treatment group was prepared in two sets of wells in duplicate. One set of cells in duplicate was incubated with the phosphorylated Akt- or ERK-specific antibody to determine phosphorylated Akt and ERK. The other set of cells in duplicate was incubated with the pan-Akt- or ERK-specific antibody to determine total Akt and ERK. Appropriate controls such as cells with no treatment or the cells with no primary but with secondary antibodies were also included. Cell culture medium was removed and fixed with 100 µl of 4% cell fixing buffer for 20 min at room temperature. Blocking buffer was removed, and the washings were performed. Cells were incubated with a primary antibody followed by a secondary antibody incubation as per the manufacturer's instructions. The reaction was developed, and the absorbance was measured at 450 nm using an ELISA plate reader.

Furthermore, the relative cell number was calculated to determine phosphorylated and total protein activity per cell number. The solution was removed from the 96-well plates, washed, and then incubated with 100 µl of cell-staining buffer for 30 min at room temperature. The washings were repeated and the cells were incubated with 1% SDS for 1 h at room temperature. The absorbance was read at 595 nm using an ELISA plate reader. To calculate the protein activity per cell number, the antibody absorbance reading at optical density (OD) 450 was divided by cell number OD595. The final relative extent of the target protein phosphorylation for Akt and ERK was calculated from the normalized phosphospecific antibody OD450-to-OD595 ratio to the pan-specific antibody OD450-to-OD595 ratio for the same experimental conditions.

Western blot analysis. Following the treatments in the different groups, the H9c2 cells were washed with PBS, enzymatically dissociated with the use of trypsin-EDTA (GIBCO), and finally collected in modified radioimmunoprecipitation assay buffer containing 50 mM Tris·HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 2 mM sodium orthovandate, 5 mM sodium fluoride, 1 mM PMSF, and mammalian protease inhibitor cocktail (Sigma). Cells were allowed to lyse following an incubation for 30 min on ice. The cell lysates were centrifuged for 15 min at 14,000 g at 4°C. The protein concentration was measured in the supernatant using a Bio-Rad protein assay. Samples containing equal amounts of proteins were subjected to 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad). The blots were incubated with the primary antibody against phosphorylated Akt and ERK (Santa Cruz) and then with secondary antibody horseradish peroxidase anti-mouse/rabbit IgG followed by detection with the chemiluminescence system. The same Western blots were stripped using stripping solution and then incubated with the primary antibody against total Akt and ERK (Santa Cruz). Following the washings, the blots were incubated with secondary antibody horseradish peroxidase anti-mouse/rabbit IgG followed by detection with the chemiluminescence system.

Data analysis. The significance of differences between values was assessed using the EXCEL program t-test. All values were expressed as means ± SE. Statistical significance was assigned when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cell death induced by a 2-h exposure of H2O2 (400 µM) in H9c2 cells replaced with fresh cell culture medium or ES-CM demonstrated morphological changes as shown in Fig. 1. Quantitative data in Fig. 2A show the decrease in cell survival (P < 0.05) following the treatment with H2O2, and this decrease was significantly improved with ES-CM. The addition of Akt inhibitor LY-294002 in ES-CM blocks the protective effects of ES-CM, suggesting that ES-CM-mediated cell survival involves the Akt pathway. In contrast, ES-CM treated with the ERK inhibitor PD-98059 did not block the cell survival effects (Fig. 2A). Furthermore, we determined the cell survival by using additional trypan blue staining. We demonstrated a decrease in cell survival following treatment with H2O2, and this decrease was inhibited with the ES-CM treatment group (Fig. 2B, P < 0.05). ES-CM + LY-294002 abolished the protective effects of ES-CM (Fig. 2B, P < 0.05). In contrast, PD-98059 shows no effect on ES-CM-protected cell proliferation (Fig. 2B). Moreover, LY-294002 alone further inhibit H2O2 decreased cell survival; however, no such effect was observed with PD-98059.


Figure 1
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  		Fig. 1. Effects of embryonic stem cells-conditioned medium (ES-CM) on H9c2 cell viability after exposure to H2O2. A: untreated H9c2 cells in control medium for 24 h. B: H9c2 cells exposed to 400 µm H2O2 for 2 h and then placed in normal cell culture control medium for 24 h. C: H9c2 cells exposed to H2O2 for 2 h and then placed in ES-CM medium for 24 h (x20).

 

Figure 2
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  		Fig. 2. Effects of ES-CM, Akt inhibitor LY-294002 (LY, 40 µM) and ERK inhibitor PD-98050 (PD, 25 µM) on H9c2 cell survival and proliferation after exposure to H2O2. A: quantitative number of H9c2 cells. Data are from the set of 7–9 independent experiments. B: quantitative percent cell viable using trypan blue method (see MATERIALS AND METHODS). Data are from the set of 8–10 independent experiments. C, control. *P < 0.05 vs. H2O2; @P < 0.05 vs. ES-CM; **P < 0.05 vs. CM + LY; #P < nonsignificant (NS) vs. CM and CM + PD.

 
Using TUNEL staining, we determined the effect of ES-CM, ES-CM + LY-294002 and ES-CM + PD-98059 on H2O2-induced apoptosis in H9c2 cells. Figure 3, A–E, demonstrates the presence of the total nuclei stained with DAPI, and Fig. 3, F–J, shows TUNEL-stained nuclei. Merged nuclei are shown in Fig. 3, K–O. The treatment of H9c2 cells with H2O2 for 24 h increases ~22% apoptosis (Fig. 3A, P < 0.05) compared with the control media group. The percentage of apoptotic nuclei induced by H2O2 was significantly reduced (P < 0.05) following treatment with ES-CM (Fig. 4A). The addition of the LY-294002 in ES-CM blocks the ES-CM-inhibited apoptosis (Fig. 4A, P < 0.05), returning the apoptotic levels to nonsignificant levels compared with the cells seen in H2O2-treated group. In contrast, PD-98059 demonstrated no effect on reduced apoptosis (Fig. 4A). The quantitative apoptosis measured by the ELISA assay was significantly reduced with ES-CM, and the reduced apoptosis was blocked with LY-294002 at a concentration of 40 and 80 µm but not with 20 µm (Fig. 4C, P < 0.05). Moreover, PD-98059 at the concentration of 12.5 and 25 µm did not block ES-CM-inhibited apoptosis (Fig. 4D). Furthermore, we determined caspase-3 activity since this is an important indicator of apoptosis and plays a major role in stress-induced apoptosis in H9c2 cells. Our data show that caspase-3 activity was higher in H2O2-induced apoptosis in H9c2 cells, and this increase was significantly inhibited by ES-CM (Fig. 4B, P < 0.05). Inhibited caspase-3 activity with ES-CM was blocked with Akt but not with ERK inhibitor (Fig. 4B), suggesting the role of Akt pathway.


Figure 3
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  		Fig. 3. Effects of ES-CM, Akt inhibitor LY (40 µM), and ERK inhibitor PD (25 µM) on H9c2 cells apoptosis after exposure to H2O2. Representative photomicrographs of total nuclei stained with 4',6-diamidino-2-phenylindole in blue (A–E), terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL)-stained nuclei in red (F–J), and merged (K–O) (x20). +ve, Positive.

 

Figure 4
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  		Fig. 4. Effects of ES-CM, Akt inhibitor LY (40 µM), and ERK inhibitor PD (25 µM) on TUNEL-positive nuclei (A), caspase-3 activity (B), and cell-death ELISA (C and D) after exposure to H2O2. A: LY, but not PD, inhibits ES-CM-reduced apoptosis, confirmed with TUNEL-positive apoptotic nuclei (n = 4–6 fields/well in each condition). Data are from the set of 5 to 6 independent duplicate experiments. B: LY, but not PD, blocks ES-CM-reduced caspase-3 activity. Data are from the set of 8–10 independent experiments. C: Akt inhibitor LY inhibits ES-CM-decreased apoptosis measured by quantitative apoptotic ELISA (see MATERIALS AND METHODS). Data are from the set of 8–10 independent experiments. D: ERK inhibitor PD shows no effect on ES-CM-decreased apoptosis measured by quantitative apoptotic ELISA (see MATERIALS AND METHODS). Data are from the set of 8–10 independent experiments. *P < 0.05 vs. H2O2; @P < 0.05 vs. ES-CM, #P < NS vs. ES-CM.

 
We next determined pAkt and pERK activities in this cell culture model and the effects of LY-294002 and PD-98050. Figure 5A shows significant decreased levels of pAkt in the H2O2 treatment compared with the control media group. Treatment with ES-CM increased pAkt levels (Fig. 5A, P < 0.05). The addition of LY-294002 (40 µm) in ES-CM demonstrated the inhibition of increased phosphorylated levels (Fig. 5A, P < 0.05). In contrast, pERK was unchanged with and without H2O2 treatment as well as following treatments with ES-CM and ES-CM + PD-98059 compared with the control media group (Fig. 5B). Furthermore, we confirmed our ELISA data using additional Western blot analysis. Figure 5C shows that the treatment with ES-CM increases pAkt levels (Fig. 5A, P < 0.05). The addition of LY-294002 (40 µm) in ES-CM demonstrated the inhibition of increased pAkt levels. In contrast, pERK was unchanged in all the groups (Fig. 5D).


Figure 5
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  		Fig. 5. Effects of ES-CM, Akt inhibitor LY (40 µM), and ERK inhibitor PD (25 µM) on phosphorylated (p)Akt and pERK activity of H9c2 cells after exposure to H2O2. A: quantitative pAkt examined by case ELISA kit. Data are from the set of 5 to 6 independent experiments. *P < 0.05 vs. control, **P < 0.05 vs. H2O2, #P < NS vs. ES-CM. B: quantitative pERK examined by case ELISA kit. Data are from the set of 5 to 6 independent experiments. #P < NS vs. H2O2 and ES-CM. C: representative Western blot analysis of pAkt. D: Western blot analysis of pERK from different groups. The same stripped membrane shows total Akt and ERK (C, bottom, and D, bottom). The data are from the sets of 3 to 4 independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Cardiac myocyte apoptosis and necrosis are present in the myocardial infarction that leads to cardiac remodeling and eventually chronic heart failure (2, 4, 5, 21). Oxidative stress is reported to be a key mediator of apoptosis in the infarcted and reperfused hearts (21, 23). Using a cell culture model system, many investigators have shown that H2O2 induces stress-mediated apoptosis in isolated adult cardiomyocytes as well as in H9c2 cells (21–23, 37). We demonstrate in this study that the treatment of H9c2 cells with H2O2 induces significant cell death. Using three independent methods such as TUNEL staining, cell death ELISA, and caspase-3 activity, we present data that establish that the cell death observed in the H9c2 cells is apoptotic. We also demonstrated that ES-CM inhibits H2O2-induced apoptosis in H9c2 cells. These findings confirm the recently published observations from our laboratory (42) using this oxidative stress-induced apoptosis model. Our laboratory (42) has also shown that four major antiapoptotic factors, including osteopontin, clusterin, cystatin-c, and tissue inhibitor of metalloproteinase 1, were present in the ES-CM that plays a role to inhibit H9c2 cell-induced apoptosis. These factors have been shown to be antiapoptotic in cancer cells, neuroblastoma cells, PC12 cells, H9c2 cells, and endothelial cells (19, 26, 30, 35, 47). However, the mechanisms of ES-CM-inhibited H9c2 cells apoptosis remain elusive.

PI3k/Akt and ERK pathways are commonly involved in the stress-induced apoptosis in in vitro and in vivo models (1, 11, 18, 25, 31). In fact, the Akt pathway is activated in the H2O2-induced apoptosis in H9c2 cells (18), HL-1 cardiomyocytes (10), as well as in in vivo models of ischemia-reperfusion injury (2, 21). In the present study, we demonstrated that pAkt promotes cell survival in the oxidative stress-induced apoptosis in H9c2 cells following treatment with ES-CM. Specifically, we noted the reduced levels of apoptosis in H9c2 cells treated with ES-CM compared with the significant increased levels of pAkt. In contrast, the increased levels of H9c2-cell apoptosis were associated with the decreased levels of pAkt (Figs. 4 and 5A). Therefore, we suggest that the dynamics of pAkt were consistent with its role as a negative regulator of H9c2-cell apoptosis in this model. Furthermore, the Akt inhibitor blocks the antiapoptotic effects of ES-CM on H9c2-cell apoptosis confirmed with TUNEL staining, apoptotic ELISA, and caspase-3 activities, suggesting that Akt activation is important and required for the prosurvival function of ES-CM under oxidative stress conditions. Our data are consistent with the recent findings that the Akt pathway mediates angiopoietin-1 (45), ghrelin (6) inhibited stress-induced apoptosis in H9c2 cells, and insulin-like growth factor-1 (IGF-1) inhibited stress-induced apoptosis in dorsal root ganglion cells (25). Importantly, it has been shown that serum deprivation induces apoptosis in different cell culture models (7, 36). With the use of different inhibitors of apoptosis, it has been demonstrated that apoptosis is mediated through the pAkt and pERK pathways (7, 8, 36). However, future studies are needed to develop a serum deprivation-induced apoptotic H9c2 cell culture model to determine the effects of ES-CM on apoptosis as well as their mechanisms.

ERK is an important MAPK family protein that plays a critical role in the cell survival, proliferation, and differentiation in many cell types, including cardiac myocytes (11, 24, 37, 46). We explored the role of ERK protein in the ES-CM-mediated cell protection in H9c2 cells. In the present study, we demonstrate there was no significant difference in the levels of pERK with and without H2O2 treatment. We also show that ES-CM was unable to upregulate ERK activity. Moreover, the ERK inhibitor PD-98050 shows no effect on ES-CM-inhibited apoptosis and ERK phosphorylation. Based on this data, we suggest that the ERK pathway is not involved in the inhibition of apoptosis with ES-CM under oxidative stress-induced circumstances.

In conclusion, our data for first time suggest that ES-CM is protective for H2O2-induced apoptosis in the H9c2 cells mediated through the Akt but not the ERK pathway. Our data are in agreement with previously published data on the IGF-II inhibition of mammary epithelial cell apoptosis that involves the Akt but not the ERK pathway (33). Moreover, we have shown that ES-CM contains four different antiapoptotic proteins (42). Therefore, the current study opens new avenues to understand the effects of each individual antiapoptotic protein using various apoptotic in vitro and in vivo models to inhibit apoptosis and its mechanisms.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
We acknowledge support provided by an American Heart Association Scientist Development Grant 0430227N and National Heart, Lung, and Blood Institute Grants 1R21-HL-085795-01A1 and 1R01-HL-090646-01 (to D. K. Singla).


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. K. Singla, Burnett's School of Biomedical Sciences, Univ. of Central Florida, 4000 Central Florida Blvd., Orlando, FL, 32816 (e-mail: dsingla{at}mail.ucf.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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