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Shin, Jeon, Kwak, Song, Lee, Jeong, Choi, Kim, Park, and Choi: Microenvironments and Cellular Proliferation Affected by Oxygen Concentration in Non-Small Cell Lung Cancer Cell Line
Original Article
Tuberculosis and Respiratory Diseases

Tuberculosis and Respiratory Diseases 2007; 63(3): 242-250.

Published online: 30 September 2007

DOI: https://doi.org/10.4046/trd.2007.63.3.242

Microenvironments and Cellular Proliferation Affected by Oxygen Concentration in Non-Small Cell Lung Cancer Cell Line

Jong Wook Shin, M.D., Eun Ju Jeon, M.D., Hee Won Kwak, M.D., Ju Han Song, M.D., Young Woo Lee, M.D., Jae Woo Jeong, M.D., Jae Cheol Choi, M.D., Jae-Yeol Kim, M.D., In Won Park, M.D., Byoung Whui Choi, M.D.

Division of Allergy, Respiratory and Critical Care Medicine, Department of Internal Medicine, Chung Ang University College of Medicine, Seoul, Korea.

Address for correspondence: In Won Park, M.D. Division of Allergy, Respiratory and Critical Care Medicine, Department of Internal Medicine, Chung-Ang University Hospital, 224-1, Huksuk-dong, Dongjak-gu, 156-755, Seoul, Korea. Phone: 82-2-6299-1401, Fax: 82-2-6263-2184, iwpark@cau.ac.kr, basthma@hanmail.net

Received 30 May 2007       Accepted 14 September 2007

Copyright © 2007 The Korean Academy of Tuberculosis and Respiratory Diseases

Abstract

Background

Abnormal angiogenesis can induce hypoxia within a highly proliferating tumor mass, and these hypoxic conditions can in turn create clinical problems, such as resistance to chemotherapy. However, the mechanism by which hypoxia induces these changes has not yet been determined. Therefore, this study was conducted to determine how hypoxia induces changes in cell viability and extracellular microenvironments in an in vitro culture system using non-small cell lung cancer cells.

Methods

The non-small cell lung cancer cell line, A549 was cultured in DMEM or RPMI-1640 media that contained fetal bovine serum. A decrease in the oxygen tension of the media that contained the culture was then induced in a hypoxia microchamber using a CO2-N2 gas mixture. A gas analysis and an MTT assay were then conducted.

Results

(1) The decrease in oxygen tension was checked the anaerobic gas mixture for 30 min and then reoxygenation was induced by adding a 5% CO2-room air gas mixture to the chamber. (2) Purging with the anaerobic gas mixture was found to decrease the further oxygen tension of cell culture media. (3) The low oxygen tension resulted in a low pH, lactic acidosis and a decreased glucose concentration in the media. (4) The decrease in glucose concentration that was observed as a result of hypoxia was markedly different when different types of media were evaluated. (5) The decrease in oxygen tension inhibited proliferation of A549 cells.

Conclusion

These data suggests that tumor hypoxia is associated with acidosis and hypoglycemia, which have been implicated in the development of resistance to chemotherapy and radiotherapy.

Keywords: Non-small cell lung cancer, Hypoxia, Oxygen, A549, Microenvironment

Figures and Tables

Figure 1
Equipments for treating different oxygen concentration in cell culture system. (A) Two 10-cm cell culture dishes are located within MIC-101 hypoxic microchamber. (B) MIC-101 hypoxic chamber with cell culture dishes is located within 5% CO2 cell culture incubator.
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Figure 2
Measurement of oxygen and carbon dioxide tension and microenvironmental components (*p<0.05). (A) Partial pressure of O2; In sixteen hours after treatment of 5% CO2-95% N2 gas mixture, O2 was decreased. (B) Partial pressure of CO2; In sixteen hours after treatment of 5% CO2-95% N2 gas mixture, CO2 was not decreased in comparison to normoxic control. (C) Glucose concentration was decreased significantly after 16 hour -treatment of hypoxia in contrast to normoxia (control). (D) The pH in hypoxia was not different from normoxic control in 16 hours of experiments. (E) Lactic acid concentration was significantly further increasing in 16 hours of hypoxic condition than normoxia. (F-I) [Na+], [K+], [Cl-], [Ca2+] were not different between normoxia and hypoxia in 16 hours of experiments.
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Figure 3
Change of partial fraction of oxygen resulted from purging anoxic gas into microchamber. After purging,partial pressure of oxygen was more drcreased from oxgen concentration of hypoxic condition without purging. (First bar; normoxia, 2nd Bar; Hypoxia without purging, 3rd Bar; Hypxoia after purging)
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Figure 4
Change of microenvironments in culture media during hypoxia-and-reoxygenation procedure. According to pre-hypoxic, post-hypoxic treatment and reoxygenation procedure, changes in partial pressure of O2 (A), CO2 (B), glucose (C) and lactic acid (D) were seen. Electrolytes were not changed significantly (E).
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Figure 5
Differential effect on glucose level according to types of cell culture media (DMEM, RPMI) (*p<0.05).
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Figure 6
Cell proliferation according to different oxygen concentration. In 48 hours, hypoxic cells proliferated more slowly than cells in normoxic condition.(―: Normoxia, ―: Hypoxia)
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