Establishment and Characterization of a Topotecan Resistant Non-small Cell Lung Cancer NCI-H460/TPT10 Cell Line

Abstract

While topotecan (TPT) is a first- and second-line chemotherapeutic drug in treating lung cancer, the development of drug resistance in tumors still reserves as a major obstacle to chemotherapeutic success. Therefore, a better understanding of the mechanisms of topotecan resistance is critical. In this study, the first topotecan-resistant human non-small cell lung cancer (NSCLC) cell line, termed NCI-H460/TPT10, was established from the parental NCI-H460 cell line. NCI-H460/TPT10 cells exhibited a 394.7-fold resistance to TPT, and cross-resistance to SN-38, mitoxantrone, and doxorubicin, compared to parental NCI-H460 cells. Overexpression of ABCG2 localized on the cell membrane, but not ABCB1 or ABCC1, was found in NCI-H460/TPT10 cells, indicating that ABCG2 was likely to be involved in topotecan-resistance. This was confirmed by the abolishment of drug resistance in NCI-H460/TPT10 cells after ABCG2 knockout. Moreover, the involvement of functional ABCG2 as a drug efflux pump conferring multidrug resistance (MDR) was indicated by low intracellular accumulation of TPT in NCI-H460/TPT10 cells, and the reversal effects by ABCG2 inhibitor Ko143. The NCI-H460/TPT10 cell line and its parental cell line can be useful for drug screening and developing targeted strategies to overcome ABCG2-mediated MDR in NSCLC.

Keywords: ABCG2; NCI-H460/TPT10; multidrug resistance; non-small cell lung cancer; topotecan.

FIGURE 1

Protein expression profile of NCI-H460/TPT10 and parental NCI-H460 cells. Western blot on the expression levels of (A) ABCG2, (B) topoisomerase I, (C) ABCB1, and (D) ABCC1 in NCI-H460 and NCI-H460/TPT10 cells. HEK293/pcDNA3.1, HEK293/ABCG2, HEK293/ABCB1, and HEK293/ABCC1 cell lines served as negative or positive controls for ABCB1, ABCG2, and ABCC1 expression, respectively. The adopted loading control was GAPDH.

FIGURE 2

Immunofluorescence microscopic images of NCI-H460 and NCI-H460/TPT10 cells. ABCG2 and DAPI fluorescence micrographs were combined to create a merged image. ABCG2 expression was shown by green fluorescence; cell nuclei were stained blue by DAPI. This experiment has been done with triplicate wells in replicated independent tests.

FIGURE 3

Abolishment of Drug Resistance by Ko143 and ABCG2 gene Knockout in NCI-H460/TPT10 Cells. Cell viability was determined by MTT assay and displayed the changes in response to different concentrations of (A) topotecan, (B) SN-38, (C) mitoxantrone, and (D) cisplatin in drug resistant NCI-H460/TPT10 and the parental NCI-H460 cells, with or without 3 μM Ko143, and in NCI-H460/TPT10 ABCG2 knockout (ko) cells as well as the vector control subline. Data points with error bars represented the mean viability (%) ± SD of at least three independent experiments, each done in triplicate. Statistical analysis was performed to compare the IC₅₀ values. * in green: p < 0.05 NCI-H460/TPT10 with Ko143 3 μM versus NCI-H460/TPT10 without Ko143. * in pink: p < 0.05 NCI-H460/TPT10-ABCG2 ko versus NCI-H460/TPT10 vector control.

FIGURE 4

Topotecan accumulation in NCI-H460 and NCI-H460/TPT10 cells. (A) Flow cytometry detection of intracellular accumulation of topotecan in cells after 2-h exposure to 100 μM topotecan with or without 2-h pretreatment with 3 μM Ko143. (B) Intracellular topotecan accumulations in cells without Ko143 pretreatment are represented by the fold of fluorescence intensity. Fluorescence intensity of the accumulated topotecan in NCI-H460 cells without Ko143 was normalized to 1. Columns and error bars represented average values with SD from three independent measurements. * indicates p < 0.05 comparing resistant cell line with or without Ko143 to parental cell line with or without Ko143; # indicates p < 0.05 comparing a group with Ko143 to the corresponding cell line group without Ko143.