reperfusion injury in cultured neonatal rat cardiomyocytes

Cell Biology International 31 (2007) 1345e1352 www.elsevier.com/locate/cellbi

Effects of polyamines on apoptosis induced by simulated ischemia/reperfusion injury in cultured neonatal rat cardiomyocytes Liping Han a, Changqing Xu a,b,*, Chunming Jiang a, Hongzhu Li a, Weihua Zhang a, Yajun Zhao a, Li Zhang a, Yanqiao Zhang a, Weiming Zhao b, Baofeng Yang b a b

Department of Pathophysiology, Harbin Medical University, Harbin 150086, China Bio-pharmaceutical Key Laboratory of Heilongjiang Province, Harbin 150086, China Received 6 April 2007; revised 5 May 2007; accepted 12 May 2007

Abstract We incubated neonatal SpragueeDawley rat cardiomyocytes in primary culture in a medium simulating ischemia (consisting of hypoxia plus serum deprivation) for 2 h, then re-incubated them for 24 h in normal culture medium to establish a model of simulated ischemia/reperfusion (I/R) injury. Apoptotic cell death was measured by MTT assay, TUNEL staining and flow cytometry. Morphological alterations were assessed by transmission electron microscopy, the expression of caspases-3 and -9 and Bcl-2 and the release of cytochrome c by Western blotting, and the intracellular free-calcium concentration ([Ca2þ]i) by laser confocal scanning microscopy. The results showed that pretreatment with 10 mmol/l spermine or spermidine significantly inhibited apoptosis in the I/R cells, suppressed the expression of caspases-3 and -9 and cytochrome c release, up-regulated Bcl-2 expression and decreased [Ca2þ]i. However, pretreatment with 10 mmol/l putrescine had the opposite effects. Evidence for this ‘‘double-edged’’ effect of polyamines on apoptosis in I/R-injured cardiomyocytes is presented for the first time. It may suggest a novel pharmacological target for preventing and treating cardiac ischemia/reperfusion injury. Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Polyamine; Heart; Ischemia; Reperfusion; Apoptosis

1. Introduction Ischemia/reperfusion (I/R) injury is a major cause of morbidity and mortality in diseases such as stroke, myocardial infarction and acute renal tubular necrosis. In I/R-injured cells, there are serial alterations in cellular metabolism and toxic molecules are generated, as indicated by the presence of necrotic and apoptotic areas in the affected organs (Bonventre, 1993; Zweier et al., 1993; Rabb et al., 1998). Apoptosis of cardiac myocytes has been recognized as a cellular mechanism of ischemia/reperfusion injury in the heart.

* Corresponding author at: Department of Pathophysiology, Harbin Medical University, Baojian Road, Harbin 150086, China. Tel.: þ86 451 8667 4548; fax: þ86 451 8750 3325. E-mail address: [email protected] (C. Xu).

A class of molecules related to cell death is represented by the polyamines spermidine, spermine and putrescine (Wallace et al., 2003). These substances belong to a family of lowmolecular-weight organic polycations that are classically known to be important mediators of cell growth, division and proliferation (Wallace et al., 2003; Ackermann et al., 2003). There is also increasing evidence that polyamines are tightly connected with apoptotic cell death in a quite complex interplay (Nikolaus and Francis, 2005). They appear to be Janus-faced regulators of cellular fate, promoting either cell proliferation or cell death depending on the cell type and on environmental signals (Thomas and Thomas, 2001). In several cell types, high levels of polyamines may directly cause cell death (Poulin et al., 1995; Stefanelli et al., 1998; Erez et al., 2002). Nothing, however, is known about the potential role of polyamines in programmed cell death after cardiac accident, specifically in I/R-mediated apoptosis.

1065-6995/$ - see front matter Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2007.05.015

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In this study, we simulated ischemia/reperfusion injury in neonatal rat cardiac myocytes in primary culture and evaluated the effects of exogenous polyamines on this model of apoptosis. The results showed that spermine and spermidine promoted the resistance of cells to the simulated I/R injury, prevented the release of cytochrome c from mitochondria and the downstream caspase activation, and inhibited intracellular calcium increase; but putrescine had the opposite effects or no effect.

1996). In brief, extracts were mixed with 10 nmol/l internal standard (1,6-hexanediamine), treated with benzoyl chloride and extracted with chloroform. The derivatives were separated on a Waters ODS C18 column (250 mm  4.6 mm, 5 mm; Waters Corporation, USA) and eluted with methanol and distilled water (65:35 V:V) at 40  C; elution was monitored by a ultraviolet detector at 229 nm (SPD-66A, Shimadzu, Japan). Polyamines were measured by HPLC (LC-6A, Shimadzu, Japan) and the polyamine concentration was expressed in nanomoles per 106 cells.

2.5. Cell viability assay 2. Materials and methods The study was approved by the Institutional Animal Research Committee and all animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health (NIH publication 86-23, revised 1986).

2.1. Materials Putrescine, spermidine and spermine were purchased from Sigma (USA); deoxygenation reagent from Japan; terminal deoxynucleotidyl transferasemediated dUTP nick-end labeling (TUNEL) kit from Roche (Germany); and anti-caspase-3, caspase-9, Bcl-2 and cytochrome c antibodies from Santa Cruz (Europe).

2.2. Cell culture and treatment

Cell viability was determined by the MTT assay. Cells were cultured in 96well plates, MTT was added to each well under sterile conditions (with a final concentration of 0.5 mg/ml), and the plates were incubated for 4 h at 37  C. Supernatant was removed and dimethylsulfoxide (150 ml/well) was added. The plates were then agitated on a plate shaker. The absorbance of each well was measured at 490 nm with a Bio-Rad automated EIA Analyzer. The viability of control cells was taken as 100%, and the others were expressed as percentages of control.

2.6. TUNEL staining Apoptosis was detected by the terminal transferase-mediated dUTP nick end-labeling (TUNEL) method. In accordance with the manufacturer’s protocol, cells in 24-well plates were fixed with 4% paraformaldehyde for 30 min at room temperature. After two washes in PBS, they were permeabilized with 0.1% Triton X-100 for 2 min at room temperature, then incubated with 50 ml TUNEL reaction mixture for 60 min at 37  C. Diaminobenzidine was used to generate an insoluble colored substrate at the site of DNA fragmentation. Finally, the cells were counterstained with methyl green for morphological evaluation and characterization of normal and apoptotic cells. All cells were analyzed under a microscope. The percentage of apoptotic cells was calculated as the ratio of the number of TUNEL-positive cells to the total number of cells, counted in three different random fields.

Primary cultures of neonatal rat cardiomyocytes were prepared by the method described previously (Chun et al., 2004). Briefly, single cells were dissociated from minced hearts of 1e2 day old Wistar rats with a 0.25% solution of crude trypsin and then cultured as monolayers at a density of 5  104 cells/ cm2 in DMEM medium equilibrated with humidified air containing 5% CO2 at 37  C. The medium contained 10% calf serum and 2 mM fluorodeoxyuridine, the latter to prevent proliferation of non-myocytes. Three days after the cells were seeded, the cultured cardiomyocytes were randomly divided into five groups. Control group: cardiomyocytes were continuously cultured for 26 h in normal culture medium. Ischemia/reperfusion (I/R) group: cells were subjected to 2 h simulated ischemia followed by 24 h of reperfusion. Spermine, spermidine and purescine groups: 10 mM spermine, spermidine or putrescine was added to the culture medium at the beginning of reperfusion. Drugs were dissolved in pre-warmed medium and added directly to the culture. For controls, equivalent volumes of medium were added. Only cultures that consisted of >95% actin-positive cells, determined by counting 300 cells in 3 different fields, were used in the analyses.

The apoptotic ratio was measured by flow cytometry as described previously (Sun et al., 2006). According to the manufacturer’s protocol, cells were washed three times with ice-cold PBS and stained with Annexin V-FITC for 15 min at room temperature in 200 ml binding buffer. Then 300 ml binding buffer was added, the cells were stained with propidium iodide for 30 min at 4  C and their fluorescence was analyzed by flow cytometry. The percentage of apoptotic cells was determined using ModFit LT software.

2.3. Simulated ischemia/reperfusion

2.8. Transmission electron microscopy

The experimental protocol used to simulate I/R was a modified version of the method proposed by Rakhit et al. (2000). Briefly, serum-containing incubation medium was replaced with serum-free DMEM 2 h before the start of the experiment. The cells were then treated with ischemic buffer solution (in mM: 118 NaCl, 24 NaHCO3, 1 NaH2PO4 $ H2O, 2.5 CaCl2 $ 2H2O, 0.5 sodium EDTA $ 2H2O, 20 sodium lactate, 16 KCl, pH 6.2). After pre-gassing with 95% N2 and 5% CO2 for at least 5 min, the ischemia buffer was added to the cells, which were then placed in a sealed chamber containing the deoxygenation reagent. The catalytic reaction of the reagent resulted in the consumption of O2 and the production of CO2. This Anaero-Pack System provided near-anaerobic conditions with an O2 concentration of <1% and a CO2 concentration of about 5% within 1 h of incubation at 37  C. The cells were exposed to these conditions for 2 h, then incubated again in glucose-containing DMEM at 37  C in 95% air and 5% CO2 (reperfusion) for 24 h.

Cells were harvested and fixed with 3.0% glutaldehyde and 1.5% paraldehyde, washed in PBS, fixed in osmium tetroxide, dehydrated in an ethanol series, embedded in Epoxy Resin, then examined under a transmission electron microscope (JEM-2000EX).

2.4. Measurement of intracellular polyamine levels

Cells were explored to 24 h of reperfusion following 2 h of ischemia and intracellular polyamines determined as described in the Methods section. Polyamine levels are nmol/106 cells. All values are means  SEM. *P < 0.05, **P < 0.01 vs control group.

At the end of the experiment, the cells were harvested. Dansylation and extraction of polyamines was based on published methods (Seiler et al.,

2.7. Flow cytometric assay

Table 1 Effect of I/R on intracellular polyamines levels Treatment

Putrescine

Spermidine

Spermine

Control I/R Pu þI/R Spd þI/R Sp þI/R

0.46  0.17 0.71  0.19* 0.84  0.23** 0.69  0.16* 0.68  0.15*

1.58  0.24 1.16  0.21** 1.17  0.20** 1.56  0.23 1.17  0.21*

2.09  0.27 1.21  0.23** 1.24  0.24** 1.25  0.26** 1.99  0.26

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B

A

*

** *

/R Pu +I

I/R +

I/R Sp d

+ Sp

C

C

I/R

on tr o

l

I/R +

I/R

*

**

Pu

+ Sp

d

+

I/R

I/R Sp

Co n

tr ol

*

* *

Pu

+

I/R

I/R Sp

d

+

+

I/R

I/R Sp

C

on

tr

ol

**

D

Fig. 1. Effect of polyamines on apoptosis of neonatal cardiomyocytes exposed to simulated ischemia/reperfusion. Cardiomyocytes were subjected to 2 h simulated ischemia, consisting of hypoxia plus serum deprivation, then re-incubated under control conditions in the presence or absence of 10 mM spermine, spermidine or putrescine for 24 h. Cells were then collected for analysis. Data are means  SEM of five determinations. ::P < 0.01 vs control; *P < 0.05; **P < 0.01 vs I/R. (A) MTTassay for cell viability. (B) DNA fragmentation as determined by the quantitative assay of apoptotic cardiomyocytes (TUNEL staining). (C) & (D) Flow cytometric analysis.

2.9. Western blot analysis of Procaspases-3 and -9 and Bcl-2

2.10. Detection of cytochrome c release from mitochondria

Total proteins were prepared from the neonatal rat myocytes as described previously (Rajeswaria and Pande, 2006). To each lane of a 10% SDS-polyacrylamide gel, 20 mg total protein was applied, electrophoresed, and transferred to a PVDF membrane. Membranes were blocked using Tris-buffered saline with 5% non-fat milk. Blots were then probed with rabbit monoclonal anti-rat caspases-3, -9 or Bcl-2 antibodies in blocking buffer, and subsequently by a secondary anti-mouse IgG antibody. Actin expression was used as control. The volumes of the protein bands were quantified by a Bio-Rad Chemi DocÔ EQ densitometer and Bio-Rad Quantity One software (Bio-Rad laboratories, Hercules, USA).

To quantify cytochrome c release, Western blot analysis of cytochrome c in the cytosolic fraction was performed as previously described (Zhang et al., 2006). Briefly, cells were collected, washed twice with ice-cold PBS and incubated in ice-cold Tris-sucrose buffer (0.35 M sucrose, 10 mM TriseHCl, pH 7.5, 1 mM ethylenediaminetetra-acetic acid, 0.5 mM dithiothreitol, 0.1 mM phenylmethanesulfonyl fluoride). After 40 min incubation they were centrifuged at 1000  g for 5 min at 4  C and the supernatant was further centrifuged at 40,000  g for 30 min at 4  C. The supernatant was retained as the cytosolic fraction. It was analyzed by Western blotting with

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Fig. 2. Ultrastructural changes in cardiomyocytes. (A) Control group; (B) Ischemia/reperfusion (I/R) group; (C) Spermine þI/R group; (D) Spermidine þI/R group; and (E) Putrescine þI/R group (15,000). In the I/R and putrescine þI/R groups, nuclear chromatin margination, aggregation and condension, and dissolution and disappearance of mitochondrial cristae, could be observed. There was no significant change in nuclear chromatin in the control, spermine or spermidine groups.

a primary rat anti-cytochrome c monoclonal antibody and a secondary goat-anti-rat IgG (Promega). Actin expression was used as control.

2.11. Measurement of [Ca2þ]i Cultured cardiomyocytes in 96-well plates were loaded with 10 mM Fluo3/AM for 60 min at 37  C, then rinsed with Ca2þ-free PBS three times to remove the extracellular Fluo-3/AM; 200 ml of normal DMEM medium was added. Excitation was set at 488 nm and emission was monitored at 530 nm. Fluorescence images indicating [Ca2þ]i were recorded using a laser confocal scanning microscope (Leica, Germany). The loaded cells were randomly divided into four groups. I/R group: cells were incubated for 8 min with normal DMEM medium and for 60 min with simulated ischemia solution, then reperfused for 30 min with DMEM medium. Spermine, spermidine and putrescine groups: the protocol was similar to I/R group except that 10 mmol/l spermine, spermidine or putrescine was added to the medium at the beginning of reperfusion.

2.12. Statistical analysis All data were obtained from at least three independent experiments that were replicated two or four times for each condition. Data are expressed as means  SEM. Comparisons among the groups were performed using KruskaleWallis two-way ANOVA. Values of P < 0.05 were considered statistically significant.

3. Results 3.1. Effect of I/R on intracellular polyamine levels To investigate whether intracellular polyamine levels were altered by the induction of I/R injury, intracellular putrescine,

spermidine and spermine levels were measured before and after the administration of exogenous polyamines. The concentrations of spermidine and spermine were significantly decreased in comparison with the control group (P < 0.01), while putrescine concentration was increased (P < 0.05), after 24 h of reperfusion following 2 h of simulated ischemia. Administration of each exogenous spermidine and spermine also restored the corresponding intracellular level to normal, but did not alter the intracellular levels of other polyamines (Table 1).

3.2. Apoptotic cell death MTT analysis showed that the viability of spermine- or spermidine-treated I/R cells was significantly greater than that of untreated or putrescine-treated I/R cells (Fig. 1A). Similarly, only 6  0.3% TUNEL positive nuclei were detected in control cells at the end of the experiment, while I/R significantly increased the number of apoptotic cells to 19  0.8% (P < 0.01 vs control). Spermine and spermidine reduced the numbers of TUNEL positive cells to 13  0.7% and 14  0.8%, respectively (P < 0.01 and P < 0.05 vs I/R), while putrescine increased them to 23  1.4% (P < 0.05 vs I/R) (Fig. 1B). Flow cytometric analysis showed similar results. Very little apoptosis was detected in control cultured cardiomyocytes. Cells underwent significant apoptosis when exposed to I/R, and this was markedly aggravated by putrescine. However, pretreatment with spermine and spermidine decreased the apoptosis rate significantly (P < 0.01 and P < 0.05, respectively, vs I/R) (Fig. 1C).

L. Han et al. / Cell Biology International 31 (2007) 1345e1352

E Caspase-3 expression

A

1349

3.5

*

3 2.5 2

** **

1.5 1 0.5

F Caspase-9 expression

B

I/R + Pu

Sp d

Sp

+

+

I/R

I/R

I/R

Co nt ro l

0

3

*

2.5 2

**

**

1.5 1 0.5

G Bcl-2 expression

C

I/R

I/R Sp

Pu

d

+

+

I/R +

I/R Sp

Co

nt

ro

l

0

2

**

*

1.5

*

1 0.5

H Cytochrome c release

D

I/R +

Sp

Pu

d

Sp

+

+

I/R

I/R

I/R

Co

nt

ro

l

0

*

3 2.5 * 2

**

1.5 1 0.5 I/R Pu

+

I/R +

I/R Sp d

+

I/R Sp

Co

nt

ro l

0

Fig. 3. Expression of pro-caspases-3 and -9 and Bcl-2 and release of cytochrome c determined by Western blotting in cardiomyocytes exposed to simulated I/R. (AeD) Representative photographs of pro-caspases-3 and -9 and Bcl-2 expression and cytochrome c release. (EeH) The intensity of each band was quantified by densitometry, and the data were normalized using the actin signal. The control group was considered the basal level (designated 1), and the other groups were expressed as fold changes compared to the control. The fold change values are means  SEM. (::P < 0.01 vs control; *P < 0.05, **P < 0.01, vs I/R).

3.3. Morphological characterization of cardiomyocyte apoptosis We examined the morphological changes associated with apoptosis in cardiomyocytes by transmission electron

microscopy. We observed margination, aggregation and condensation of the nuclear chromatin, and dissolution and disappearance of the mitochondrial cristae, in the I/R and putrescine groups. However, there was no evident histopathological change in the spermine and spermidine groups (Fig. 2).

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1350

A

Before ischemia

Ischemia

Ischemia/reperfusion

Non-treated

Spermine

Spermidine

Putrescine

4500

B

Non-treated 4000

Spermine Spermidine

Fluorescent intensity

3500

Putrescine

3000

* **

*

2500

**

2000 1500 1000 500

s 80

s

s

80

58

52

s

80 46

s

80

80

40

80

s

s 34

28

s 22

80

s 80

0s

80 16

10

48

0s 24

0s

0

Fig. 4. Changes of intracellular free calcium concentration in neonatal cardiomyocytes subjected to simulated ischemia/reperfusion. (A) Fluorescent images respectively represent cardiomyocytes grown in normal DMEM medium, incubated in simulated ischemia solution and re-incubated in normal DMEM medium in the presence or absence of 10 mM spermine, spermidine or putrescine. (B) The changes in fluorescent intensities indicating intracellular Ca2þ were recorded continuously by laser scanning confocal microscopy under different treatment regimes. Intracellular Ca2þ was recorded for 98 min in total.

3.4. Expression of caspases-3 and -9 and release of cytochrome c To investigate the pathway through which polyamines activate or suppress apoptosis, we analyzed the expression

of procaspases-3 and -9 and the release of cytochrome c by Western blotting. Compared with control cells, the expression of procaspases-3 and -9 and the release of cytochrome c were significantly increased in I/R cells (P < 0.01), and putrescine enhanced this increase (P < 0.05 vs I/R group). Spermine and

L. Han et al. / Cell Biology International 31 (2007) 1345e1352

spermidine, however, down-regulated the expression of the pro-caspases and inhibited cytochrome c release (P < 0.05 or P < 0.01 vs I/R group) (Fig. 3). 3.5. Expression of Bcl-2 Spermine and spermidine significantly up-regulated the expression of Bcl-2 (P < 0.05 or P < 0.01 vs I/R group), but putrescine decreased it (P < 0.05 vs I/R group) (Fig. 3). 3.6. Measurement of intracellular calcium concentration ([Ca2þ]i) We continuously examined [Ca2þ]i by laser confocal scanning microscopy. The fluorescence intensity peak increased during reperfusion (P < 0.01 vs normal condition). When spermine or spermidine was pre-added to the reperfusion medium, the peak decreased (P < 0.01 and P < 0.05 respectively vs I/R group), while putrescine had little effect (Fig. 4). 4. Discussion Very little is known about the involvement of polyamines in the execution of apoptosis in cardiac myocytes. In the present work, we examined whether these polycations play a role in the apoptosis of neonatal rat cardiomyocytes in a model of simulated ischemia/reperfusion (I/R) injury. The results showed that I/R caused disturbance of polyamine metabolism: spermine and spermidine decreased while putrescine increased. Then we administered exogenous polyamines and evaluated apoptosis by the TUNEL method, flow cytometry and cell viability assays using MTT. The apoptosis rate was increased in I/R cells; putrescine enhanced this rate while spermine and spermidine decreased it. To investigate the pathway through which polyamines activate or suppress apoptosis, we analyzed the expression of procaspases-3 and -9. Activation of the caspase family is known to be a crucial mechanism for inducting apoptotic death signals. Caspases participate in a cascade that is triggered in response to pro-apoptotic signals and culminates in the cleavage of a set of proteins, resulting in disassembly of the cell (Enari et al., 1998; Thornberry and Lazebnik, 1998). Caspase-3 is the primary activator of apoptotic DNA fragmentation (Wolf et al., 1999). Our study indicated that I/R procaspase-3 and caspase-9 while spermine and spermidine decreased their expression, but putrescine had the opposite effect. This indicates that the suppression or induction of apoptosis by polyamines is mediated by a caspase-dependent pathway. To determine whether polyamines affect the mitochondrial pathway, we studied Bcl-2 expression, release of cytochrome c and intracellular calcium concentration ([Ca2þ]i). The antiapoptotic protein Bcl-2 resides in the outer mitochondrial membrane and inhibits cytochrome c release (Hattori et al., 2001). Recent studies have demonstrated that when the intracellular Ca2þ concentration is elevated, much larger amounts of Ca2þ can accumulate in the mitochondria (Schanne et al.,

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1979; Ott et al., 2002), and this can alter the outer mitochondrial membrane permeability leading to the release of pro-apoptotic mitochondrial proteins such as cytochrome c and apoptosis-inducing factor (Cory and Adams, 2002; Kowaltowski et al., 2000). Our study demonstrated that the quantities of cytochrome c in the cytoplasm of I/R cells was significantly higher than that in the control group, but spermine and spermidine significantly up-regulated Bcl-2 and inhibited cytochrome c release. In addition, [Ca2þ]i was markedly increased during simulated I/R, while spermine and spermdine significantly inhibited this increase. Putrescine, however, had no such effect. These results indicate that spermine and spermidine may also suppress apoptosis through the mitochondrial pathway. The effect of putrescine may differ from that of spermine and spermidine because of differences in chemical structure and spatial configuration. In conclusion, polyamines (spermine, spermidine and putrescine) are a ‘‘double-edged sword’’ for myocardial cells, and their pro- and anti-apoptotic effects may be related to differences in covalent bonds and conformation. Our study suggests that regulation of polyamine metabolism is a potential novel approach for the treatment of myocardial injury associated with apoptosis. Acknowledgements This research is supported by project grants from the National Natural Science Foundation of China (No. 30370577 and No. 30470688) and the Postgraduate Innovation Foundation of Heilongjiang Province. References Ackermann JM, Pegg AE, McCloskey DE. Drugs affecting the cell cycle via actions on the polyamine metabolic pathway. Prog Cell Cycle Res 2003;5:461e8. Bonventre JV. Mechanisms of ischemic acute renal failure. Kidney Int 1993;43:1160e78. Chun ZG, Meng XF, Zhua ZH, Liu JS, Deng AG. Connective tissue growth factor regulates the key events in tubular epithelial to myofibroblast transition in vitro. Cell Biol Int 2004;28:863e73. Cory S, Adams JM. The Bcl-2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2002;2:647e56. Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata SA. A caspase-activated DNase that degrades DNA during apoptosis and its inhibitor ICAD. Nature 1998;391:43e50. Erez O, Goldstaub D, Friedman J, Kahana C. Putrescine activates oxidative stress dependent apoptotic death in ornithine decarboxylase overproducing mouse myeloma cells. Exp Cell Res 2002;281:148e56. Hattori R, Hernandez TE, Zhu L, Maulik N, Otani H, Kaneda Y, et al. An essential role of the antioxidant gene Bcl-2 in myocardial adaptation to ischemia: an insight with antisense Bcl-2 therapy. Antioxid Redox Signal 2001;3:403e13. Kowaltowski AJ, Vercesi AE, Fiskum G. Bcl-2 prevents mitochondrial permeability transition and cytochrome c releasing via maintence of reduced pyridine nucleotides. Cell Death Differ 2000;7:903e10. Nikolaus S, Francis R. Polyamines and apoptosis. J Cell Mol 2005;9:623e42. Ott M, Roberson JD, Gogvadze V, Zhiovotovsky B, Orrenius S. Cytochrome c release from mitochondria proceeds via a two-step process. Proc Natl Acad Sci USA 2002;99:1259e63.

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L. Han et al. / Cell Biology International 31 (2007) 1345e1352

Poulin R, Pelletier G, Pegg AE. Induction of apoptosis by excessive polyamine accumulation in ornithine decarboxylase-overproducing L1210 cells. Biochem J 1995;311:723e7. Rabb H, Haq M, Shull GE, Soleimani M. Possible molecular basis for changes in potassium handling in acute renal failure. J Am Soc Nephrol 1998;9: 605e13. Rajeswaria J, Pande G. Direct association between caspase 3 and a5b1 integrin and its role during anoikis of rat fibroblasts. Cell Biol Int 2006;30: 963e9. Rakhit RD, Edwards RJ, Mockridge JW, Baydoun AR, Wyatt AW, Mann GE, et al. Nitric oxide-induced cardioprotection in cultured rat ventricular myocytes. Am J Physiol 2000;278:H1211e7. Schanne FAX, Kane AB, Young EE, Farber JL. Calcium dependence of toxic cell death. Science 1979;206:700e2. Seiler N, Delcros JG, Vaultier M, Le Roch N, Havouis R, Douaud F, et al. Bis (7-amino-4-azaheptyl) dimethylsilane and Bis (7-ethylamino-4-azaheptyl) dimethylsilane: inhibition of tumor growth in vitro and in vivo. Cancer Res 1996;56:5624e30. Stefanelli C, Bonavita F, Stanic’ I, Mignani M, Facchini A, Pignatti C, et al. Spermine causes caspase activation in leukaemia cells. FEBS Lett 1998; 437:233e6.

Sun YH, Liu MN, Li H, Shi S, Zhao YJ, Wang R, et al. Calcium-sensing receptor induces rat neonatal ventricular cardiomyocyte apoptosis. Biochem Biophys Res Commun 2006;350:942e8. Thomas T, Thomas TJ. Polyamines in cell growth and cell death: molecular mechanisms and therapeutic applications. Cell Mol Life Sci 2001;58: 244e58. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science 1998;281: 1312e6. Wallace HM, Fraser AV, Hughes A. A perspective of polyamine metabolism. Biochem J 2003;376:1e14. Wolf BB, Schuler M, Echeverri F, Green DR. Caspase-3 is the primary activator of apoptotic DNA fragmentation via DNA fragmentation factor-45/inhibitor of caspase-activated DNase inactivation. J Biol Chem 1999;274: 30651e6. Zhang WH, Fu SB, Lu FH, Wu B, Gong DM, Pan ZW, et al. Involvement of calcium-sensing receptor in ischemia/reperfusion-induced apoptosis in rat cardiomyocytes. Biochem Biophys Res Commun 2006;347:872e81. Zweier JL, Duilio C, Kuppusamy P, Santoro G, Elia PP, Tritto I, et al. A short burst of oxygen radicals at reflow induces sustained release of oxidized glutathione from postischemic hearts. J Biol Chem 1993;268: 18532e41.