Role of ABCG2 as a biomarker for predicting resistance to CPT-11/SN-38 in lung cancer

Abstract

To examine the mechanism of resistance to 7-ethyl-10-hydroxycamptothecin (SN-38) in lung cancer, we continuously exposed the non-small-cell lung cancer (NSCLC) cell line NCI-H23 to SN-38 and selected the SN-38-resistant clone H23/SN-38. After 2 months of culturing in SN-38-free conditions, H23/SN-38 cells recovered their sensitivity to SN-38 and were subsequently established as the revertant H23/SN-38REV cell line. Because H23/SN-38 cells show cross resistance to certain anticancer drugs, such as topotecan, etoposide, doxorubicin and mitoxantrone, we examined the gene and protein expression levels of drug efflux transporters of the ATP-binding cassette (ABC) family. We found that both gene and protein expression of ABCG2/BCRP (ABCG2) in H23/SN-38 cells was increased compared with that in NCI-H23 cells and H23/SN-38REV cells. The cellular accumulation of topotecan in H23/SN-38 cells was decreased compared with that in NCI-H23 and H23/SN-38REV cells, and treatment with reserpine (an inhibitor of ABCG2) increased the cellular accumulation of topotecan in H23/SN-38 cells. Furthermore, treatment with reserpine also altered the sensitivity of H23/SN-38 cells to SN-38. These results indicate that the upregulation of ABCG2 was functional, and related to the resistance of H23/SN-38 cells to SN-38. Moreover, we found that gene expression levels of ABCG2 were significantly correlated with the concentration of SN-38 for 50% cell survival in 13 NSCLC cells (r=0.592, P<0.05). The present results indicate that the induction of ABCG2 by SN-38 does confer acquired resistance to CPT-11/SN-38, but the induction of ABCG2 and subsequent drug resistance are reversible. However, the expression level of ABCG2 may be a useful indicator of CPT-11/SN-38 activity in lung cancer.

Figure 1

(A) Gene expression levels of ABCG2 and ABCC1 in NCI‐H23, H23/SN‐38 and H23/SN‐38REV cells. (B) Protein expression levels of ABCG2 in NCI‐H23, H23/SN‐38 and H23/SN‐38REV cells. (C) Gene expression levels of ABCG2 in PC‐6, PC‐6/SN2‐5 and PC‐6/SN2–5REV cells.

Figure 2

Flow cytometry analysis of the cellular accumulation of topotecan. The flow cytometric assay was carried out just after a 4 h‐exposure to 200 µM topotecan. Uptake of topotecan was measured using a fluorescence‐activated cell sorter. The presence of topotecan is shown by a single line, and absence is shown by a shaded line. A fluorescence peak shift to the right was observed in NCI‐H23 cells. In H23/SN‐38 cells, a fluorescence peak shift was not observed in the absence of reserpine.

Figure 3

(A) Pretreatment with the ABCG2 inhibitor reserpine affects the SN‐38 cytotoxicity of H23 and H23/SN‐38 cells. The sensitivity of H23/SN‐38 cells to SN‐38 was increased by 30‐min pretreatment with 20 µM reserpine, whereas that of the parental NCI‐H23 cells did not change.

Figure 4

(A) Relationship between the basal expression levels of the ABCG2 gene by real‐time polymerase chain reaction and SN‐38 sensitivity in 13 NSCLC cell lines. (B) Gene and protein expression levels of ABCG2 in 13 NSCLC cell lines: 1, NCI‐H23; 2, RERF‐LC‐AI; 3, SK‐LC‐1; 4, ACC‐LC‐176; 5, RERF‐LC‐MT; 6, QG56; 7, RERF‐LC‐MS; 8, RERF‐LC‐OK; 9, PC‐9; 10, ACC‐LC‐94; 11, ACC‐LC‐174; 12, SK‐LC‐6; and 13, PC‐10. (C) Comparison of the drug concentration for 50% cell survival (IC₅₀) values for SN‐38 between ABCG2‐expressed and ABCG2‐not expressed NSCLC cells by western blot.