Korean J Physiol Pharmacol.  2012 Feb;16(1):11-16. 10.4196/kjpp.2012.16.1.11.

Effect of 5-FU and MTX on the Expression of Drug-resistance Related Cancer Stem Cell Markers in Non-small Cell Lung Cancer Cells

• Yi H
• Cho HJ
• Cho SM
• Jo K
• Park JA
• Lee SH
• Chang BJ
• Kim JS
• Shin HC

• KMID: 2285434
• DOI: http://doi.org/10.4196/kjpp.2012.16.1.11

Abstract

Cancer stem cells (CSCs) are often characterized by the elevated expression of drug-resistance related stem-cell surface markers, such as CD133 and ABCG2. Recently, we reported that CSCs have a high level of expression of the IL-6 receptor (IL-6R). The purpose of this study was to investigate the effect of anticancer drugs on the expression of the drug resistance-related cancer stem cell markers, ABCG2, IL-6R, and CD133 in non-small cell lung cancer (NSCLC) cell lines. A549, H460, and H23 NSCLC cell lines were treated with the anticancer drugs 5-fluorouracil (5-FU; 25 microg/ml) and methotrexate (MTX; 50 microg/ml), and the expression of putative CSC markers was analyzed by fluorescent activated cell sorter (FACS) and the gene expression level of abcg2, il-6r and cd133 by reverse transcriptasepolymerase chain reaction (RT-PCR). We found that the fraction of ABCG2-positive(+) cells was significantly increased by treatment with both 5-FU and MTX in NSCLC cells, and the elevation of abcg2, il-6r and cd133 expressions in response to these drugs was also confirmed using RT-PCR. Also, the number of IL-6R(+) cells was increased by MTX in the 3 cell lines mentioned and increased by 5-FU in the H460 cell line. The number of CD133(+) cells was also significantly increased by both 5-FU and MTX treatment in all of the cell lines tested. These results indicate that 5-FU and MTX considerably enhance the expression of drug-resistance related CSC markers in NSCLC cell lines. Thus, we suggest that antimetabolite cancer drugs, such as 5-FU and MTX, can lead to the propagation of CSCs through altering the expression of CSC markers.

Keyword

Cancer stem cell; 5-FU/MTX; ABCG2/IL-6R/CD133; Drug resistance

MeSH Terms

Carcinoma, Non-Small-Cell Lung
Cell Line
Drug Resistance
Fluorouracil
Gene Expression
Methotrexate
Neoplastic Stem Cells
Receptors, Interleukin-6
Fluorouracil
Methotrexate
Receptors, Interleukin-6

Figure

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  Fig. 1 Analysis of ABCG2 expression after 5-FU or MTX treatment. A549, H460, and H23 non-small cell lung cancer cell lines were treated with 5-FU (25 µg/ml) or MTX (50 µg/ml) for 24 hours and incubated with a PE-anti-ABCG2 antibody. The number of fluorescent-conjugated cells in 104 cells was analyzed by fluorescence activated cell sorter (FACS).

• 

  Fig. 2 Analysis of IL-6R expression after 5-FU or MTX treatment. A549, H460, and H23 non-small cell lung cancer cell lines were treated by 5-FU (25 µg/ml) or MTX (50 µg/ml) for 24 hours and incubated with a FITC-anti-IL-6R antibody. The number of fluorescent-conjugated cells in 104 cells was analyzed by fluorescence activating cell sorter (FACS).

• 

  Fig. 3 Analysis of CD133 expression after 5-FU or MTX treatment. A549, H460, and H23 non-small cell lung cancer cell lines were treated with 5-FU (25 µg/ml) or MTX (50 µg/ml) for 24 hours and incubated with a PE-anti-CD133 antibody. The number of fluorescent-conjugated cells in 104 cells was analyzed by fluorescence activating cell sorter (FACS).

• 

  Fig. 4 Gene expression of abcg2 after 5-FU or MTX treatment. (A) A549, H460, and H23 non-small cell lung cancer cell lines were treated with 5-FU (25 µg/ml) or MTX (50 µg/ml) for 6, 12, and 48 hr. After the incubation period, total RNA was extracted, and mRNA levels were analyzed by RT-PCR. PCR amplification was performed using specific primer pairs designed from published human gene sequences. A housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (gapdh), was selected as an internal control. (B) The densities of the DNA bands of abcg2 were analyzed by Image J Software (NIH, http://rsb.info.nih.gov/nih_image/index.html).

• 

  Fig. 5 Gene expression of IL-6R after 5-FU or MTX treatment. (A) A549, H460, and H23 non-small cell lung cancer cell lines were treated with 5-FU (25 µg/ml) or MTX (50 µg/ml) for 6, 12, and 48 hr. After the incubation period, total RNA was extracted, and mRNA levels were analyzed by RT-PCR. PCR amplification was performed using specific primer pairs designed from published human gene sequences. A housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (gapdh), was selected as an internal control. (B) The densities of the DNA bands of il-6r were analyzed by the Image J Software.

• 

  Fig. 6 Gene expression of CD133 after 5-FU or MTX treatment. (A) A549, H460, and H23 non-small cell lung cancer cell lines were treated with 5-FU (25 µg/ml) or MTX (50 µg/ml) for 12 hr. After the incubation period, total RNA was extracted, and mRNA levels were analyzed by RT-PCR. PCR amplification was performed using specific primer pairs designed from published human gene sequences. A housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (gapdh), was selected as an internal control. (B) The densities of the DNA bands of cd133 were analyzed by the Image J Software.

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