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Abstract
Purpose
In a previous study, we have shown that anticancer agents inhibiting topoisomerases improve survival of tumor cells under hypoxic condition. In the present study, we evaluated whether and how cell survival effect of the anticancer agents under hypoxic conditions could be eliminated by the addition of nitroimidazoles, a class of bioreductive agents.
Methods
Human hepatocellular carcinoma cells (HepG2) were incubated with different combinations of pimonidazole (1~1,000 µg/ml) and doxorubicin (0.1 or 1 µg/ml) concentrations under different O2 concentrations [1, 3, 5, 10 and 21 O2]. Then cell numbers, glucose concentrations and lactic acid concentrations in the medium were measured, and DNA fragmentation assay was performed. Finally, different combinations of nitroimidazoles, such as pimonidazole, misonidazole, etanidazole, tinidazole, metronidazole, ornidazole or dimetridazole, and anticancer agents, such as doxorubicin, campothecin, epirubicin, dactinomycin, etoposide or mitomycin C was added to the cell culture medium under hypoxic conditions (1% O2).
Results
Pimonidazole at a concentration of 100 µg/ml eliminated cell survival effect of doxorubicin at the concentrations of 0.1 and 1 µg/ml under hypoxic condition (1% O2) by promoting apoptosis. Almost all the cells died even after 24 hours of incubation for all the oxygen concentrations at a combination of 100 µg/ml pimonidazole and 1 µg/ml doxorubicin. Finally, pimonidazole at a concentration of 100 µg/ml, and misonidazole or etanidazole at a concentration of 1,000 µg/ml eliminated cell survival effect of all the anticancer agents tested under hypoxic condition.
Figures and Tables
Fig. 1
Effect of pimonidazole concentrations on the cell viability under hypoxic conditions. Human hepatocellular carcinoma cells (HepG2) were grown in 4 ml of MEM culture medium at 2.5×105 cells/60 mm culture dish under normoxic condition for 48 hours before transferred to fresh culture medium with different pimonidazole concentrations under hypoxic conditions. Cell viability (A), glucose concentrations (B) and lactic acid concentrations in the medium (C) were measured during cell culture. The Y axis, Ratio, in (A) indicates the number of viable cells at a specific culture day divided by the number of viable cells at day zero. Error bars represent the standard deviation of at least three samples taken from a single run. ‡, † and * represent P<0.001, P<0.01 and P<0.05, respectively.
Fig. 2
Effect of pimonidazole concentrations on the cell viability under normoxic conditions. Human hepatocellular carcinoma cells (HepG2) were grown in 4 ml of MEM culture medium at 2.5×105 cells/60 mm culture dish under normoxic condition for 48 hours before transferred to fresh culture medium with different pimonidazole concentrations under normoxic conditions. Cell viability (A), glucose concentrations (B) and lactic acid concentrations in the medium (C) were measured during cell culture. The Y axis, Ratio, in (A) indicates the number of viable cells at a specific culture day divided by the number of viable cells at day zero. Error bars represent the standard deviation of at least three samples taken from a single run. ‡, † and * represent P<0.001, P<0.01 and P<0.05, respectively.
Fig. 3
Effect of pimonidazole concentrations on the cell viability in the presence of doxorubicin under hypoxic conditions. Human hepatocellular carcinoma cells (HepG2) were grown in 4 ml of MEM culture medium at 2.5×105 cells/60 mm culture dish under normoxic condition for 48 hours before transferred to fresh culture medium with different combinations of pimonidazole and doxorubicin concentrations under hypoxic conditions. Cell viability (A), glucose concentrations (B) and lactic acid concentrations in the medium (C) were measured during cell culture. The Y axis, Ratio, in (A) indicates the number of viable cells at a specific culture day divided by the number of viable cells at day zero. Error bars represent the standard deviation of at least three samples taken from a single run. ‡, † and * represent P<0.001, P<0.01 and P<0.05, respectively.
Fig. 4
Effect of pimonidazoe on the DNA fragmentation in the presence of doxorubicin under hypoxic conditions. Human hepatocellular carcinoma cells (HepG2) were grown in 4 ml of MEM culture medium at 2.5×105 cells/60 mm culture dish under normoxic condition for 48 hours before transferred to fresh culture medium with 100 µg/ml of pimonidazole in the presence of 0 (A), 0.1 (B) and 1 µg/ml (C) doxorubicin concentrations under hypoxic conditions. At the indicated times, all the cells in the 60 mm dishes were lysed, and chromosomal DNA was taken and loaded to 1.5% agarose gel. Lane M (100 bp DNA marker), Lane 1 (0), Lane 2 (12), Lane 3 (24), Lane 4 (30), Lane 5 (36), Lane 6 (48), Lane 7 (72) hours of culture under normoxic condition).
Fig. 5
Effect of pimonidazole concentrations on the cell viability in the presence of doxorubicin under normoxic conditions. Human hepatocellular carcinoma cells (HepG2) were grown in 4 ml of MEM culture medium at 2.5×105 cells/60 mm culture dish under normoxic condition for 48 hours before transferred to fresh culture medium with different combinations of pimonidazole and doxorubicin concentrations under normoxic conditions. Cell viability (A), glucose concentrations (B) and lactic acid concentrations in the medium (C) were measured during cell culture. The Y axis, Ratio, in (A) indicates the number of viable cells at a specific culture day divided by the number of viable cells at day zero. Error bars represent the standard deviation of at least three samples taken from a single run. ‡, † and * represent P<0.001, P<0.01 and P<0.05, respectively.
Fig. 6
Effect of pimonidazole on the DNA fragmentation in the presence of doxorubicin under normoxic conditions. Human hepatocellular carcinoma cells (HepG2) were grown in 4 ml of MEM culture medium at 2.5×105 cells/60 mm culture dish under normoxic condition for 48 hours before transferred to fresh culture medium with 100 µg/ml of pimonidazole in the presence of 0 (A), 0.1 (B) and 1 µg/ml (C) doxorubicin concentrations under normoxic conditions. At the indicated times, all the cells in the 60 mm dishes were lysed, and chromosomal DNA was taken and loaded to 1.5% agarose gel. Lane M (100 bp DNA marker), Lane 1 (0), Lane 2 (12), Lane 3 (24), Lane 4 (30), Lane 5 (36), Lane 6 (48), Lane 7 (72) hours of culture under normoxic condition.
Table 1
Effect of oxygen concentrations on the cell viability in the presence of different combinations of pimonidazole and doxorubicin concentrations
Table 2
Effect of nitroimidazoles on the cell viability in the presence of different anticancer agents
*concentrations (µg/ml) of anticancer agents where the agents showed improvement of cell viability the most under hypoxic conditions (1% oxygen concentration); †concentrations (µg/ml) of nitroimidazole where improvement of cell viability by the respective anticancer agents was eliminated; ‡numbers in the parenthesis of each column represent incubation time (hours) where the nitroimidazole effect occurs.
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