Mitoxantrone resistance in a small cell lung cancer cell line is associated with ABCA2 upregulation

Abstract

The aim of this study was to find factors that could explain the accumulation difference of mitoxantrone in the BCRP1-negative GLC4-MITO cell line compared to GLC4. Comparative genomic hybridisation (CGH) was applied to determine chromosomal differences between GLC4 and GLC4-MITO. Comparative genomic hybridisation analysis revealed gain of 2q, 6p, 9q, 13q, 14q, 15q, 19q and Xp and loss of 1p, 2q, 3p, 3q, 4q, 6q, 8q, 11p, 16p, 17q, 18p, 20p and Xq. In the over-represented chromosomal areas, seven transporter genes were identified: ABCB6, ABCB2 (TAP1), ABCB3 (TAP2), ABCF1 (ABC50), ABCC10 (MRP7), ABCA2 (ABC2) and ABCC4 (MRP4). No RNA or protein upregulation was observed for ABCB6, ABCF1, ABCC10, ABCC4, ABCB2 and ABCB3, but an increased expression was detected for ABCA2 mRNA in GLC4-MITO. ABCA2 is known to be involved in resistance to estramustine. In the MTT assay, GLC4-MITO was two-fold resistant to estramustine compared to GLC4. Coincubation with estramustine and mitoxantrone increased mitoxantrone accumulation in GLC4-MITO, while this was not affected in GLC4. This suggests that estramustine is able to block mitoxantrone efflux in GLC4-MITO cells. These data reveal that cellular reduction of mitoxantrone in a mitoxantrone-resistant cell line is associated with overexpression of ABCA2.

Figure 1

Comparative genomic hybridisation profiles of GLC4-MITO vs GLC4 and normal control DNA. (A) CGH profile of GLC4-MITO vs GLC4 (blue line) (B) CGH profile of GLC4 vs normal male DNA. (C) CGH profile of GLC4-MITO vs normal male DNA. (D) Explanation of schematic representation of a CGH profile gain >1.15(green bar) loss <0.85 (red bar).

Figure 2

RT–PCR of selected efflux pump genes. (A, B): Expression of MRP1 MRP2 MRP5 BCRP β -2 microglobulin and HRTP mRNA in the mitoxantrone-resistant cell line GLC4-MITO and sensitive cell line GLC4. (C): Expression of ABCB6 ABCF1 ABCC10 ABCC4 ABCA2 β 2-microglobulin and HRTP in the mitoxantrone-resistant cell line GLC4-MITO and its sensitive parent line GLC4.

Figure 3

Representative cytotoxity profile of the estramustine sensitivities of GLC4-MITO (▪), GLC4-ADR (□) and GLC4 () measured with an MTA. Values represent the means of four experiments±s.d.

Figure 4

Effect of estramustine on mitoxantrone drug accumulation in GLC4 and GLC4-MITO. In total, 0, 10 and 25 μM estramustine were added together with 3 μM mitoxantrone to 1 × 10⁶ GLC4 and GLC4-MITO cells. Median fluorescence intensity was measured indicating the relative amount of mitoxantrone accumulation. Values represent the median mitoxantrone fluorescence relative to GLC4 with 3 μM mitoxantrone. Values represent the means of three experiments±s.d. No significant difference was observed in GLC4 with or without estramustine.