J Korean Surg Soc.  2009 Jun;76(6):337-347. 10.4174/jkss.2009.76.6.337.

Effect of Combination of Anticancer Agents and Nitroimidazoles on the Survival of Human Hepatocellular Carcinoma Cells under Hypoxic Conditions

• Lim SH
• Lee JY
• Park SH
• Kim YH
• Suh HS
• Park JB
• Lee J

• KMID: 1464948
• DOI: http://doi.org/10.4174/jkss.2009.76.6.337

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 microg/ml) and doxorubicin (0.1 or 1 microg/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 microg/ml eliminated cell survival effect of doxorubicin at the concentrations of 0.1 and 1 microg/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 microg/ml pimonidazole and 1 microg/ml doxorubicin. Finally, pimonidazole at a concentration of 100 microg/ml, and misonidazole or etanidazole at a concentration of 1,000 microg/ml eliminated cell survival effect of all the anticancer agents tested under hypoxic condition.
CONCLUSION
Combination therapy of doxorubicin (adriamycin) with pimonidazole can maximize dororubicin efficacy by eliminating cell survival effect of doxorubicin under hypoxic conditions in treating solid tumors, such as breast cancer.

Keyword

Anticancer agent; Nitroimidazole; Hypoxia; Combination therapy; Apoptosis

MeSH Terms

Anoxia
Antineoplastic Agents
Apoptosis
Breast Neoplasms
Carcinoma, Hepatocellular
Cell Count
Cell Culture Techniques
Cell Survival
Dactinomycin
Dimetridazole
DNA Fragmentation
Doxorubicin
Epirubicin
Etanidazole
Etoposide
Glucose
Humans
Lactic Acid
Metronidazole
Misonidazole
Mitomycin
Nitroimidazoles
Ornidazole
Oxygen
Tinidazole
Antineoplastic Agents
Dactinomycin
Dimetridazole
Doxorubicin
Epirubicin
Etanidazole
Etoposide
Glucose
Lactic Acid
Metronidazole
Misonidazole
Mitomycin
Nitroimidazoles
Ornidazole
Oxygen
Tinidazole

Figure

• 

  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.

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