Expression Profiling of ABC Transporters in a Drug-Resistant Breast Cancer Cell Line Using AmpArray

Abstract

ATP-binding cassette (ABC) membrane proteins comprise a superfamily of transporters with a wide variety of substrates. Humans have 49 members in this superfamily. Several human ABC transporters, such as ABCB1 and ABCC1, have been attributed to cause multidrug resistance (MDR) in cancer treatment when over-expressed. In the past, an MDR cancer cell line MCF7/AdVp3000 has been selected, and overexpression of ABCG2 was thought to cause MDR in this cell line. However, ectopic overexpression of ABCG2 in MCF7 cells could not explain the high drug resistance level observed with the selected cell line. In this study, we designed an AmpArray analysis to profile whether other ABC transporters were also selected to contribute to the increased drug resistance in MCF7/AdVp3000 cells. We found that 16 ABC transporters, including ABCG2, had Ն1.5-fold altered expression in MCF7/AdVp3000 compared with the parental MCF7 cells. In particular, the expression of ABCA4 and ABCC3 was increased by 132-and 459-fold, respectively, whereas ABCG2 was increased by ϳ3000-fold. Furthermore, the elevated expression of these three transporters reversed with the reversed drug resistance phenotype, and silencing ABCC3 expression in MCF7/ AdVp3000 cells significantly reduced doxorubicin resistance. Thus, other ABC transporters in addition to ABCG2 are likely to contribute to the MDR selected in MCF7/AdVp3000 cells. This study also shows that AmpArray can be used as a quick and easy tool to profile the expression of ABC transporters in resistant cell lines and tumor samples for potential use in individualized design of therapy.Drug resistance to chemotherapy frequently occurs in cancers and is a major obstacle to successful cancer treatment. Studies with tumor cell lines have revealed that multidrug resistance (MDR) can develop and thus cause chemotherapy failure. Advances in elucidating the molecular basis of the MDR phenotype indicate that elevated membrane expression of several drug efflux pumps such as P-glycoprotein (Pgp or ABCB1), multidrug resistance protein 1 (MRP1 or ABCC1), and ABCG2 is a frequent cause of MDR in human cancers

Use of arrays to investigate the contribution of ATP-binding cassette transporters to drug resistance in cancer chemotherapy and prediction of chemosensitivity

“…Although the most frequently selected ABC transporters in various drug resistant cell lines appeared to be ABCB1 and ABCC1, consistent with their known roles in MDR, new roles in drug resistance previously unknown for some ABC transporters were also found using these profiling analyses. For example, ABCC3 and ABCC5 were both found upregulated in the Adriamycin-selected MCF7/AdVp1000 [24] and MCF7/AdVp3000 [40] cell lines using two different arrays, strongly suggesting that these transporters may contribute to Adriamycin resistance. Although these transporters may be selected by verapamil that was present www.cell-research.com | Cell Research Jian-Ting Zhang 321 npg during the selection of these cell lines, the role of ABCC3 in Adriamycin resistance has been validated functionally by the siRNA knock down strategy [40].…”

“…For example, ABCC3 and ABCC5 were both found upregulated in the Adriamycin-selected MCF7/AdVp1000 [24] and MCF7/AdVp3000 [40] cell lines using two different arrays, strongly suggesting that these transporters may contribute to Adriamycin resistance. Although these transporters may be selected by verapamil that was present www.cell-research.com | Cell Research Jian-Ting Zhang 321 npg during the selection of these cell lines, the role of ABCC3 in Adriamycin resistance has been validated functionally by the siRNA knock down strategy [40]. Clearly, further studies are still needed to more definitively demonstrate that the over-expression of these transporters causes Adriamycin resistance.…”

“…Another simpler and inexpensive AmpArray approach ( Figure 2, Table 2) has also been designed recently and tested for investigating the expression profiles of 47 ABC transporters using RT-PCR on a 96-well plate [40]. This comparative assay is quick, convenient, and economical, as 47 ABC transporters are assayed at the same time on a regular thermal cycler that houses a 96-well plate format.…”

“…Of course, simultaneous analyses of cell lines with intermediate drug resistance generated during stepwise selections and revertant cell lines that have lost resistance as demonstrated in the studies by Lamendola et al [19] and Liu et al [40] would help identify genes that likely contribute to the resistance of the cells. Further functional validation of these genes in drug resistance (Table 3) using siRNA-mediated silencing, ectopic over-expression, and known inhibitors would be very fruitful and could help verify the new drug resistance functions of these ABC transporters.…”

“…Eighty-eight such classifiers performed accurately to predict drug sensitivity. Unfortunately, no Table 1 Human ABC Transporters Subfamily Members ABCA ABCA1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A12, A13 ABCB ABCB1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11 ABCC ABCC1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13 ABCD ABCD1, D2, D3, D4 ABCE ABCE1 ABCF ABCF1, F2, F3 ABCG ABCG1, G2, G4, G5, G8 MCF7/AdVp1000 Adriamycin [24] MCF7/AdVp3000 Adriamycin [40] a The functional validation was conducted following identification by array analyses to verify the functional role of the ABC transporter in resistance to corresponding drugs tested. Some ABC transporters have known roles in resistance to the corresponding drugs from prior studies, which are not counted as functional validation in this table.…”