Praca oryginalna Original paper
The aim of the present work was to evaluate the effect of hypoxia on cardiomycyte toxicity induced by anticancer agents. Numerous studies have shown that hypoxia changes the toxic response of numerous cancer cell lines to some anticancer drugs (3, 10). This mechanism plays a key role in drug resistance observed during anticancer treatment (2). It appears reasonable that the toxicity of such drugs towards normal cells is at a higher risk when the supply of oxygen is limited. It seems probable that arteriosclerosis may efficiently reduce the flux of oxygen to cardiomyocytes, thus mod-ifying the side effects of anticancer drugs. Generally, under hypoxia conditions, the exacerbation of car-diomyocyte toxicity can be expected. On the other hand, some anticancer agents, such as doxorubicin or tirapazamine, undergo a reductive biotransformation and the atmospheric oxygen plays a crucial role in that process as the starting point of free-radical generation (4, 9, 11). Under hypoxia conditions, at a low oxygen
level, the development of oxidative stress is restricted. In that case, hypoxia may be expected to play a protec-tive role. On the other hand, in our unpublished study carried out on rats, 5-fluorouracyl caused disturbance in the cardiac redox equilibrium by changing NADH/
NAD+ and NADPH/NADP+ ratios. These changes in
the redox balance may be affected by hypoxia. In view of the above facts, it is impossible to make a prediction about the direction of changes caused by hypoxia in patients treated with anticancer drugs. In the current study the toxic effects of doxorubicin, tirapazamine, and 5-fluorouracyl on cardiomyocyte culture lines under hypoxia and normoxia conditions were tested.
Material and methods
The study was carried out on a H9c2 cell line. H9c2 is a subclonal of the original clonal cell line derived from embryonic BD1X rat heart tissue exhibiting many of the properties of the skeletal muscle. Cell culture was conduct-ed in 12-well plates in Dubecco Modificonduct-ed Eagle’s Mconduct-edium (DMEM) with addition of 10% fetal bovine serum. The
Effect of hypoxia on toxicity induced by
anticancer agents in cardiomyocyte culture
SŁAWOMIR MANDZIUK, PATRYCJA KUŚ*, JAROSŁAW DUDKA*, BARBARA MADEJ-CZERWONKA**, MONIKA CENDROWSKA-PINKOSZ**,
MAGDALENA IWAN*, IWONA ŁUSZCZEWSKA-SIERAKOWSKA***, AGNIESZKA KORGA* Department of Pneumology, Oncology and Allergology, Medical of University, Jaczewskiego 8, 20-090, Lublin, Poland
*Medical Biology Unit, Medical University of Lublin, Jaczewskiego 8, 20-090, Lublin, Poland **Departmentof Human Anatomy, Medical University of Lublin, Jaczewskiego 8, 20-090, Lublin, Poland ***Department of Animal Anatomy and Histology, University of Life Sciences, Akademicka 12, 20-950, Lublin, Poland
Received 30.10.2013 Accepted 21.01.2014
Mandziuk S., Kuś P., Dudka J., Madej-Czerwonka B., Cendrowska-Pinkosz M., Iwan M., Łuszczewska-Sierakowska I., Korga A.
Effect of hypoxia on toxicity induced by anticancer agents in cardiomyocyte culture
Summary
The main goal of the study was to determine whether hypoxia augments the toxicity of anticancer drugs towards cardiomyocytes. Drugs selected for this experiment were those that disturb the cardiac redox equilibrium. Cardiomyocytes were incubated for 24 h with doxorubicin, tirapazamine, and 5-fluorouracil, each at three doses, under normoxia and under 50% and 90% hypoxia. The cytotoxic effect was evaluated on the basis of the percentage of living cells, cell vitality (assessed by the MTT assay), and morphology. In addition, the oxidative marker and pH value were determined. Varied protective effects of hypoxia on cell morphology were observed in all cases except the medium concentration of tirapazamine. The 50% hypoxia prevented the toxic effects of all tested drugs. The 90% hypoxia, on the other hand, was effective against the cytotoxic action of doxorubicin and 5-fluoruracil, but the cytotoxicity of tirapazamine increased. It was found that under the 90% hypoxia the oxidative stress observed under normoxia and the 50% hypoxia was greatly reduced. The study revealed that the above drugs did not activate anaerobic glycolysis.
Keywords: Doxorubicn, 5-fluorouracile, tirapazmine, cardiomoyocytes H9c2
*) Publikacja dofinansowana przez Ministerstwo Nauki i Szkonictwa Wyższego w ramach programu Index Plus, nr umowy 77/JxP/dem 2012.
cells were kept in a CO2 incubator (NewAir, USA) at stable temperature (37°), CO2 concentration (5%), and humidity conditions. After reaching 70-80% confluence, the culture was passaged. The cells were incubated for 24 h with three concentrations of doxorubicin (1 µM, 5 µM and 10 µM), tirapazamine (5 µM, 25 µM and 100 µM,), and 5-fluoro-uracil (10 µM, 50 µM and 200 µM). A 24 h observation was conducted in a microscope hypoxia chamber under normoxia, 50% hypoxia (CO2: 5%, O2: 10.5%, and N2: 84.5%) and 90% hypoxia (CO2: 5%, O2: 2.1%, and N2: 92.9%) conditions. The control was prepared in the same manner, but in the absence of the agents tested. After the incubation period, the cells were evaluated in a phase-contrast microscope. The general morphology, dead cells, the extent of growth inhibition, the ability to proliferate and form colonies, vacuolisation, the presence of granularity, and the degree of cell detachment from the substrate were assessed. Additionally, the number of cells and their vital-ity were measured automatically in the presence of trypan blue, and the number of dead cells was counted with a cell counter (Invirogen, USA). Cytotoxicity was confirmed by the MTT assay. Briefly, the yellow tetrazolium (MTT) salt
is reduced by metabolically active cells, in part by the action of dehydrogenase enzymes, to generate reducing equiva-lents, such as NADH and NADPH. The resulting intracel-lular purple formazan was solubilized and quantified by spectrophotometric means with a microplate reader Power Wave xs (BioTek, USA) according to the manufacturer’s in-structions. RedoxSensor Red CC-1 and MitoTracker Green FM were used to assess the oxidative stress. MitoTracker Green FM selectively labels mitochondria regardless of the mitochondria potential. Cardiomyocytes were incubated at 3 µM RedoxSensor Red CC-1 and 0.3 µM Mitotracker Green FM at a temperature of 37°C for 20 minutes. After this period the cardiomyocytes were washed twice with PBS buffer. The results of dyeing were assessed with a fluores-cent microscope Nikon Eclipse Ti (USA). The pH value was measured with a pH-meter Sentron (Netherlands).
Results and discussion
Morphological appraisal of cardiomyocytes. The
50% hypoxia did not significantly change the morphol-ogy of cardiomyocytes compared with normoxia con-ditions (Fig. 1). There was only a slight augmentation
of the cells, which covered densely most of the surface. Under the 90% hypoxia, despite the above changes, an insignificant reduction in the cell culture density was found. The dark nuclei of cardiomyocytes and a greater number of dead cells were also observed, compared with the nor-moxia control. Under nornor-moxia conditions, doxorubicin caused significant changes in the shape and size of the cells tested. The contraction of cardiomyocytes was associated with the blebbing and swelling of the nuclei, and an increased nucleus/ cytoplasme ratio was observed. In cyto-plasm a small granulation became visible, and contact growth was inhibited. With an increasing concentration of doxorubicin, there was an increase in the number of dead cells. Interestingly, the toxic effect of the drug was smaller under the 50% hypoxia. Cardiomyocytes kept their shape and the nuclei were mostly normal. The presence of normal cells was also observed. With an increasing level of hypoxia (90%), a decreasing number of normal cells were observed, but their number was smaller than under normoxia.
As the concentration of tirapazamine increased under normoxia conditions, the drug diminished the normal cell number and weakened the cell culture density. Between some cells contact growth was still observed. Tirapazamine leads to disor-ders in the shape of cells and the nucleus/ cytoplasma ratio, as well as to the dark tint of nucleus. Under the 50% hypoxia, contrary to similar observations in the
pres-Fig. 1. The morphological changes in cardiomyocytes related to the drug used and hypoxia conditions (magnification × 150 and 200)
DOX 1 DOX 5 DOX 10 TPZ 5 TPZ 25 TPZ 100 Drug
ence of doxorubicin, the toxic effect of tirapazamine was clearer than un-der normoxia conditions. The major-ity of cells were dead and deformed. The number of dead cells increased under the 90% hypoxia. A vast ma-jority of cells were deformed, and many of them became spherical. No contact growth was found. There were bubbles and aggregates of apoptotic cells.
5-fluorouracil caused changes in morphology and a slight increase in the number of dead cells in the car-diomyocyte culture under normoxia. However, under these conditions there were no apparent differences dependent on drug concentration.
The main observations were the inhibition of contact growth, irregular line, and disruption of the nucleus.
Under the 50% hypoxia the percentage of normal cells was higher than under normoxia. Nevertheless, the substrate was less sparsely covered, and the cells maintained their shape and the nucleus/cytoplasm ratio. A similar situation was revealed under the 90% hypoxia.
Automatic evaluation of the vitality of cardio-myocytes. In the case of cardiomyocytes incubated
with doxorubicin, a vitality assay with the use of an automatic counter confirmed the results of the mi-croscopic examination (Tab. 1). The percentage of living cells incubated under 50% and 90% hypoxia increased at all doxorubicin doses tested. The same effect was observed for 5-fluorouracil. The vitality of cells incubated with tirapazamine increased under the 50% hypoxia, but under the 90% hypoxia, vitality was below the level found under normoxia.
MTT assay. Under normoxia, in agreement with the
microscopic and automatic counting assessment, the results of the MTT assay confirmed the cytotoxic effect of doxorubicin (for all concentrations), tirapazamine (for a higher concentration) and 5-fluorouracile (for the lowest and highest concentrations) (Tab. 1).
Except in the case of 5-fluorouracile at the high-est dose, the 50% hypoxia caused a protective effect against cytotoxicity observed under normoxia for all concentrations of the drugs tested. A similar effect was observed when cardiomyocytes were incubated under the 90% hypoxia. However, the highest tirapazamine concentration under the 90% hypoxia led to an
ex-tremely high cytotoxicity compared with normoxia conditions.
Assessment of pH value changes. In control
probes, the intensification of the hypoxia level caused a decrease in the pH value (Tab. 2). Under normoxia, there were no significant changes in the pH value after incubation with all drug concentrations tested. All con-centrations of the drugs improved the pH value under both the 50% and 90% hypoxia. This effect was not related to the dose.
Oxidative stress assessment. Under normoxia,
oxi-dative stress in cardiomycytes was visible at all doxo-rubicin concentrations tested, and it increased with the increasing concentration of the drug, compared with the normoxia control (Fig. 2). Under the 50% hypoxia the fluorescence signal of oxidative agents was slightly smaller than under normoxia. Under the 90% hypoxia, the oxidative signal was extremely low.
In the presence of tirapazamine under normoxia the level of the signal for oxidative stress in cardio-myocytes did not differ from the normoxia control. A noticeable increase in this signal was found at the highest concentration of tirapazamine. A similar effect was observed when cardiomyocytes were incubated under the 50% hypoxia. Under the 90% hypoxia the level of the fluorescence signal for oxidative stress was extremely low.
The level of fluorescence for 5-fluorouracil under normoxia and under the 50% hypoxia was clearly higher than in the normoxia control. Under the highest hypoxia (90%) the oxidative stress signal was noted only occasionally.
Tab. 1. The percentage of living cells measured automatically in the presence of trypan blue and the results of the MTT assay
N Concentration (µM) Vitality/MTT
Normoxia 50% Hypoxia 90% Hypoxia
Control – 62 2.088 90 2.652 72 1.952 DOX 1 53 1.732 82 2.491 87 1.754 5 54 1.700 80 2.108 79 1.801 10 78 1.699 89 1.835 80 1.795 TPZ 5 78 1.792 89 2.491 72 2.093 25 84 1.757 98 2.180 68 1.889 100 37 1.430 89 1.835 43 0.216 5-FU 10 44 1.073 88 2.036 81 1.984 50 65 1.189 86 1.919 70 1.884 200 52 1.993 100 1.851 77 1.664
Tab. 2. Changes in the pH value referring to drug action under different oxygen conditions
Control DOX (µM) TPZ (µM) 5-FU (µM)
1 5 10 5 25 100 10 50 200
Normoxia 7.7 7.7 7.6 7.7 7.7 7.6 7.7 7.6 7.7 7.6
50% Hypoxia 6.61 6.87 6.95 6.94 6.80 6.81 6.89 6.90 6.93 6.95
It was assumed that drugs that affect the redox equilibrium may exacerbate the toxic effect on normal cells with limited oxygen consumption. Cardiomyocytes are at a high risk of hypoxia because of arte-riosclerosis. The aim of this study was to determine whether hypoxia may increase the toxicity of anticancer agents towards a cardiomyocyte culture. The anticancer agents selected for the study were doxo-rubicin, tirapazamine, and 5-fluorouracil. Doxorubicin is a drug with a broad spec-trum of antitumor activity, used in therapy for over forty years (6, 7). This drug trans-fers an electron onto O2, which leads to superoxide radical formation, resulting in oxidative stress (5, 9, 11). Tirapazamine, an experimental anticancer drug, has a similar mechanism of cytotoxic effect (12), whereas 5-fluorouacil has not been reported as an agent generating oxidative stress. Our unpublished study, however, has shown that 5-fluorouracil significantly
changed the NADH/NAD+ and NADPH/
NADP+ ratios in the heart of rats, leading
to increased sensitivity to other agents af-fecting the redox equilibrium.
The present study suggests that the toxic effect of doxorubicin under normoxia con-ditions is associated with oxidative stress. Under normoxia, a decrease in cell vitality, confirmed by the MTT test and morpho-logical changes, correlated with a higher oxidative status. Under the 50% hypoxia, cell vitality, MTT and the intensity of morphology changes were not related to the oxidative status, which is clear if these results are compared with the normoxia control. Seemingly paradoxically, under the 90% hypoxia, the morphology and vitality of cells, assessed by an automatic counter and the MTT test, were protected. It is probable that a low level of O2 limits superoxide radical formation and a sec-ondary oxidative stress.
Our findings did not show any link between tirapazamine-related changes in morphology, cell vitality, and oxida-tive stress under normoxia conditions. Surprisingly, oxidative stress was visible under the 50% hypoxia. One may expect that a lower level of O2 restricts the forma-tion of superoxide radicals, thus preventing
Fig. 2. The oxidative stress in cardiomyocytes incubated with doxorubicin (DOX), tirapa-zamine (TPZ) and 5-fluorouracil (5-FU) under different oxygen condition (magnification × 150, 200, and 300)
Drug
[µM] Normoxia Hypoxia 50% Hypoxia 90%
DOX 1 DOX 5 DOX 10 TPZ 5 TPZ 25 TPZ 100 5-FU 10 5-FU 50 5-FU 200
oxidative stress. However, the absence of oxidative stress was found in cardiomyocytes incubated with tirapazamine under the 90% hypoxia.
In the case of 5-fluorouracil, oxidative stress was observed under normoxia and under the 50% hypoxia, whereas the 90% hypoxia did not cause such an effect at any of the drug concentrations tested. Because oxida-tive stress did not correlate with the vitality and toxic changes in morphology we can conclude that these changes were not closely related to oxidative stress.
The changes in the pH value were very clear for control, comparing normoxia and both hypoxia conditions. Some drugs, including doxorubicin (1, 8) and tirapazamine (13), have a toxic effect on cell mitochondria, causing disturbance in key enzymes activity. This mechanism may lead to the activation of glycolytic pathway and lactate production even under a sufficient supply of oxygen. A toxic effect of doxo-rubicin and tirapazamine has been described towards mitochondria. However, our results did not show any significant changes in the pH value under normoxia conditions for any of the concentrations of the drugs tested. At both levels of hypoxia and for all the drug concentrations tested, pH changes should be ascribed to a lower level of oxygen rather than to mitochondria disturbances.
Acknowledgement
The study was conducted with the equipment pur-chased under the project “The equipment of innovative laboratories doing research on new medicines used in the therapy of civilization and neoplastic diseases”, part of the Operational Program Development of Eastern Poland 2007-2013, Priority Axis I Modern Economy, Operations I.3 Innovation Promotion.
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Corresponding author: Sławomir Mandziuk, Department of Pneumology Oncology and Allergology, Medical University of Lublin, Jaczewskiego 8, 20-090 Lublin, Poland; e-mail: slawman7@wp.pl
