ABSTRACT
Title
Evaluation of Doxorubicin effects in Human Cardiac Progenitor Cells
Authors
R. Russo, S. Costantino, A. De Angelis, G. Esposito, E. Migliaccio, E. Piegari, K. Urbanek, L. Berrino, F. Rossi
Dept. of Experimental Medicine, Section of Pharmacology, Second University of Naples, Italy
Dept. of Experimental Medicine, Section of Pharmacology, Second University of Naples, Italy
Abstract
The anthracycline doxorubicin (DOXO) is a very effective anticancer drug. The most serious side effect of long-term DOXO treatment is cardiomyopathy followed by congestive heart failure (Singal, Iliskovic, 1998).
Cardiotoxicity of the anthracycline is not restricted to cardiomyocytes, but affects resident Cardiac Progenitors Cells (CPCs) even more dramatically. Our previous in vitro and in vivo findings indicated that, in a rat model, the determining event responsible for the initiation and evolution of the myopathy occurs at the level of the CPCs compartment. Inhibition of CPCs division in combination with accumulation of oxidative DNA damage, growth arrest, cellular senescence, and apoptosis decreased the number of functionally competent progenitors in the failing heart. The dramatic depletion of the endogenous pool of CPCs interfered with the physiological turnover of cardiomyocytes and their regeneration in the presence of diffuse cell death (De Angelis et al., 2010).
DOXO toxicity and its possible clinical relevancy were therefore assessed in human CPCs (hCPCs).
hCPCs were obtained by biopsies or by surgical waste materials, cultured in vitro and treated for 24 and 48 hours with 0.1-, 0.5-, 1- μmol/L concentration of DOXO.
Specifically our aim was to evaluate the effects of DOXO on hCPC proliferation, migration and differentiation.
MTT assay measured mitochondrial metabolic rate, documenting the dose- and time-dependent DOXO-induced decrease of viable cell number. In agreement with MTT analysis, cell proliferation detected by BrdU incorporation demonstrated that DOXO reduced the number of dividing hCPCs during S-phase.
Protein expression profiling was determined by Western Blotting. ATM kinase and phospho-p53 were upregulated in DOXO-treated hCPCs. Moreover, DOXO treatment resulted in a persistent increase in p16INK4a that coupled with the dose-dependent increase of SA-beta-gal positivity. The migratory capacity of hCPCs was markedly reduced in DOXO-treated cells in dose- and time-dependent manner.
Finally, the differentiation capacity into myogenic, smooth muscle cell and endothelial cell lineages was impaired in DOXO treated hCPCs when cultured in differentiation medium.
To evaluate whether the accumulation of an increasing population of p16 positive cells could account for a progressive and irreversible early myocardial senescence in DOXO treated patients, cells are going to be treated with DOXO for 24 and 48 hours and cultured for 7 days in fresh medium and eventually analysed. Preliminary data on hCPCs cultured for 7 days after DOXO treatment, indicated a persistent increase in p16INK4a suggesting irreversible growth arrest and cellular senescence.
DOXO treatment induced cellular senescence in hCPCs, leading to the formation of a senescent endogenous pool of hCPCs, unable to guarantee a suitable adaptive response to stress conditions. This mechanism could partially be responsible for DOXO-induced cardiotoxic effects that could develop several years after treatment in the presence of physical exercise, pregnancy and viral infections (Lefrak et al., 1973; Ali et al., 1994). The hCPCs inability to migrate after DOXO treatment could reduce their regenerative capacity, further promoting dilated cardiomyopathy progression toward heart failure.
Singal PK, Iliskovic N. N Engl J Med, 1998; 339:900-5
De Angelis A et al. Circulation 2010; 121:276-92
Lefrak EA et al. Cancer 1973;32:302-14
Ali MK et al. Cancer 1994; 74:182-8
Cardiotoxicity of the anthracycline is not restricted to cardiomyocytes, but affects resident Cardiac Progenitors Cells (CPCs) even more dramatically. Our previous in vitro and in vivo findings indicated that, in a rat model, the determining event responsible for the initiation and evolution of the myopathy occurs at the level of the CPCs compartment. Inhibition of CPCs division in combination with accumulation of oxidative DNA damage, growth arrest, cellular senescence, and apoptosis decreased the number of functionally competent progenitors in the failing heart. The dramatic depletion of the endogenous pool of CPCs interfered with the physiological turnover of cardiomyocytes and their regeneration in the presence of diffuse cell death (De Angelis et al., 2010).
DOXO toxicity and its possible clinical relevancy were therefore assessed in human CPCs (hCPCs).
hCPCs were obtained by biopsies or by surgical waste materials, cultured in vitro and treated for 24 and 48 hours with 0.1-, 0.5-, 1- μmol/L concentration of DOXO.
Specifically our aim was to evaluate the effects of DOXO on hCPC proliferation, migration and differentiation.
MTT assay measured mitochondrial metabolic rate, documenting the dose- and time-dependent DOXO-induced decrease of viable cell number. In agreement with MTT analysis, cell proliferation detected by BrdU incorporation demonstrated that DOXO reduced the number of dividing hCPCs during S-phase.
Protein expression profiling was determined by Western Blotting. ATM kinase and phospho-p53 were upregulated in DOXO-treated hCPCs. Moreover, DOXO treatment resulted in a persistent increase in p16INK4a that coupled with the dose-dependent increase of SA-beta-gal positivity. The migratory capacity of hCPCs was markedly reduced in DOXO-treated cells in dose- and time-dependent manner.
Finally, the differentiation capacity into myogenic, smooth muscle cell and endothelial cell lineages was impaired in DOXO treated hCPCs when cultured in differentiation medium.
To evaluate whether the accumulation of an increasing population of p16 positive cells could account for a progressive and irreversible early myocardial senescence in DOXO treated patients, cells are going to be treated with DOXO for 24 and 48 hours and cultured for 7 days in fresh medium and eventually analysed. Preliminary data on hCPCs cultured for 7 days after DOXO treatment, indicated a persistent increase in p16INK4a suggesting irreversible growth arrest and cellular senescence.
DOXO treatment induced cellular senescence in hCPCs, leading to the formation of a senescent endogenous pool of hCPCs, unable to guarantee a suitable adaptive response to stress conditions. This mechanism could partially be responsible for DOXO-induced cardiotoxic effects that could develop several years after treatment in the presence of physical exercise, pregnancy and viral infections (Lefrak et al., 1973; Ali et al., 1994). The hCPCs inability to migrate after DOXO treatment could reduce their regenerative capacity, further promoting dilated cardiomyopathy progression toward heart failure.
Singal PK, Iliskovic N. N Engl J Med, 1998; 339:900-5
De Angelis A et al. Circulation 2010; 121:276-92
Lefrak EA et al. Cancer 1973;32:302-14
Ali MK et al. Cancer 1994; 74:182-8