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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 2, 2006 as doi:10.1096/fj.05-4452fje. |
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* Clinica di Gastroenterologia ed Epatologia, Department Of Clinical and Experimental Medicine, University of Perugia, Perugia, Italy;
Department of Pharmaceutical Sciences, University of Padova, Padova, Italy;
Laboratorio di Patologia Vascolare, Istituto Dermopatico dell’Immacolata, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy; and
Laboratorio di Biologia Vascolare e Terapia Genica, Centro Cardiologico Fondazione Monzino, Istituto di Ricovero e Cura a Carattere Scientifico, Milan, Italy
1Correspondence: Department of Clinical and Experimental Medicine, University of Perugia, Via Enrico dal Pozzo, Perugia 06122, Italy. E-mail: lsant{at}unipg.it
SPECIFIC AIMS
The anthracyclines, such as epirubicin (EPI) and doxorubicin, are chemotherapy agents used to treat breast, liver, and colon cancers, as well as sarcomas and leukemia. Despite their potent antitumor activity, anthracycline-based chemotherapies associates with an increased risk of progressive dilatory congestive heart failure. Cellular mechanisms of EPI-induced myocardial damage involve a mitochondria-dependent apoptosis of cardiomyocytes. Polyethylene glycol is a widely used polymer for drug delivery. Conjugation with polyethylene glycol results in a reduced immunogenicity and increased hydro-solubility of the new drug compared with the parent molecule. Polyethylene glycol derivatives are more stable toward degradative enzymes and show an increased molecular weight, resulting in a prolonged half-life.
Nitric oxide (NO) is a diffusible messenger that has been involved in numerous physiological and pathological conditions. NO sensitizes neoplastic cells to ionizing radiation and photodynamic therapy and increases the antitumoral activity of a number of chemotherapy agents. Moreover, NO protects endothelial cells and cardiomyocytes from apoptosis induced by oxidative stress, proinflammatory cytokines and chemotherapy agents, suggesting that it may be useful in the prevention of anthracycline-induced cardiomyopathy.
As the polyethylene glycol backbone allows addition of multiple molecules of NO, we have taken advantage of this property to generate a series of polyethylene glycol-EPI (p-EPI) derivatives carrying different amounts of NO. We report here on the effect of one such agent obtained by adding 8 molecules of NO to p-EPI (p-EPI-NO).
PRINCIPAL FINDINGS
1. p-EPI-NO is metabolized by cancer and normal cells to generate NO
For all in vitro studies we used 3 cell lines: Caco-2, a human colon cancer cell line; H9c2, embryonic rat heart-derived myoblasts, and a primary culture of human umbilical endothelial cells (HUVEC).
Exposure of the three cell lines to p-EPI-NO resulted in a concentration- and time-dependent increase in nitrite/nitrate formation in cell supernatants, while no release of NO was observed by incubating p-EPI-NO with medium alone or exposing cells to p-EPI and EPI.
2. p-EPI-NO exerts a potent cytotoxic activity against colon cancer cells and protects cardiomyocytes and HUVEC from apoptosis
As shown in Fig. 1
A, incubation of Caco-2 cell line with p-EPI-NO resulted in a concentration-dependent induction of apoptosis that was significantly higher as compared with either EPI and p-EPI (n=8, P<0.05). Consistent with these findings, p-EPI-NO exerted a differential regulation of pro- and antiapoptotic proteins. Thus, while p-EPI-NO induced a marked up-regulation of Bax and phosphorylated p53, it caused a lower increase of Bcl-2 expression in comparison with EPI and p-EPI (Fig. 1B-D
). Apoptotic features in Caco-2 cells exposed to p-EPI-NO correlated with increased cytochrome c translocation from mitochondria to cytosol and caspase 3 activation (n=8, P<0.05).
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Caco-2 cells express high levels of survivin mRNA, a potent antiapoptotic protein that is implicated in cancer resistance against chemotherapy. Confirming previous studies, we found that exposure of Caco-2 cells to EPI and p-EPI resulted in a further increase of survivin mRNA expression. On the other hand, incubating Caco-2 cells with p-EPI-NO resulted in a reduction of survivin mRNA expression to levels significantly lower than control cells (n=8; P<0.05).
Exposure of H9c2 and HUVEC to EPI resulted in a concentration-dependent increase of apoptosis that was only partially reduced by EPI pegylation. In contrast, exposure of H9c2 and HUVEC to p-EPI-NO resulted in a percentage of cell apoptosis not significantly different from that of control cells (Fig. 2
A, B). To investigate whether p-EPI-NO protects also adult myocardial cells from EPI-induced apoptosis, cardiomyocytes isolated from adult mice were incubated up to 48 h with p-EPI and p-EPI-NO. Again, as shown in Fig. 2C, D
, we found that whereas incubation of the cells with p-EPI resulted in a marked increase in cell apoptosis, p-EPI-NO was not cytotoxic against cardiomyocytes.
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Exposure of H9c2 and HUVEC to EPI and p-EPI resulted in p53 phosphorylation and Bax up-regulation. These events were not observed in cells treated with p-EPI-NO (n=8, P<0.05). Moreover, in contrast to EPI and p-EPI, exposure of H9c2 cells and HUVEC to p-EPI-NO increased the expression of the antiapoptotic protein Bcl-2 and significantly reduced cytochrome c translocation and caspase 3 activation.
3. Effect of p-EPI-NO on energy metabolism in normal and cancer cells
As demonstrated by 
m collapse, exposure of Caco-2 cells to p-EPI and its NO derivative resulted in mitochondrial membrane damage. While EPI and p-EPI caused a similar drop in m in H9c2 cells and HUVEC, exposure of these cells to p-EPI-NO resulted in m hyperpolarization that lasted for at least 8 h.
Exposure of colon cancer cells and cardiomyocytes to EPI and p-EPI associates with a profound decline in O2 consumption and ATP generation. In contrast, although p-EPI-NO inhibited the cytochrome c oxidase activity and O2 consumption in both cell types, it caused a profound decline of ATP generation only in Caco-2 cells, while the ATP concentration remained unchanged in H9c2 cells and HUVEC for up to 8 h.
Lactate concentrations were determined as an index of glycolytic activity. Untreated Caco-2 cells generate more lactate than H9c2 cells. Incubation with p-EPI-NO increased lactate production by 2.5-fold in H9c2 cells and HUVEC but not in Caco-2 cells, suggesting that cancer cells are unable to further activate the glycolytic pathway to produce ATP.
CONCLUSIONS AND SIGNIFICANCE
The central findings of this study is that p-EPI-NO exerts a more potent cytotoxic activity against cancer cells in comparison with EPI and p-EPI, while protects endothelial cells and cardiomyocytes from anthracycline-induced injury.
Consistent with previous results, we found that EPI-induced cell death involves activation of proteins belonging to the mitochondrial pathway of apoptosis, including p53, Bax, cytochrome c, and caspase-3. In contrast to EPI and p-EPI, p-EPI-NO exerts opposite effects in normal and cancer cells. Thus, while in colon cancer cells the NO derivative of p-EPI activates p53, up-regulates Bax expression, and induces cytoplasmic translocation of cytochrome c, in endothelial cells and cardiomyocytes it did not induce p53 activation or Bax up-regulation and increased Bcl-2 expression. Overexpression of Bcl-2 could be responsible at least in part for the protective activity exerted by p-EPI-NO on H9c2 cells and HUVEC.
Exposure of cancer and normal cells to EPI and p-EPI results in mitochondrial membrane depolarization, demonstrated by the drop of m. p-EPI-NO exerts opposite effect on m in normal and cancer cells. Indeed, while the NO derivative caused a further potentiation of mitochondrial injury in Caco-2 cells, it caused m hyperpolarization in H9c2 cells and HUVEC, preventing the cytoplasmic release of cytochrome c and protecting cardiomyocytes and endothelial cells from EPI-induced cell death. We speculate that the effect on m hyperpolarization is related to the local release of NO for at least two main reasons. First, EPI and p-EPI caused a marked drop of m also in H9c2 and HUVEC; and second, NO-dependent hyperpolarization of m is a well documented phenomenon that has been correlated with cell protection from apoptotic death in other cell systems. m hyperpolarization induced by NO is related to inhibition of mitochondrial respiration at the level of cytochrome c oxidase (complex IV of the mitochondrial respiratory chain), which generates a "metabolic hypoxia," where the available O2 cannot be adequately used by the cell bioenergetic. Because O2 consumption is essential for oxidative phosphorylation, the major metabolic consequence of these interactions will be a reduction of oxidative breakdown of glucose, leading to impairment of cells ability to generate ATP. The inhibition of cellular respiration is of particular relevance in highly proliferative cells, such as tumoral cells. Indeed cancer cells maintain a high glycolytic rate even in the presence of oxygen, a phenomenon first described >70 years ago and known as the Warburg effect.
Our results provide evidence that addition of an NO-releasing moiety to pegylated EPI confers to the drug a new and unique cytotoxic profile. By dynamically regulating cell respiration and mitochondrial functions (Fig. 3
), NO increases the antitumoral activity of the chemotherapy agent in cancer cell lines, while confers protection against anthracycline-induced apoptosis of cardiomyocytes and endothelial cells. This study provides the ground for development of potent anti-cancer drugs based on the combination of NO with pegylated anthracyclines.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4452fje;
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