Purpose ABCG2 overexpression has been linked to resistance to topoisomerase inhibitors leading us to examine the potential interaction between ABCG2 and becatecarin. antibody 5D3 compared to parental cells. Increased ABCG2 expression was confirmed NOX1 by immunoblot. Conclusions Our results suggest that becatecarin is transported by ABCG2 and can induce ABCG2 expression in cancer cells. and was shown to inhibit topoisomerase I and II but its poor water solubility precluded its clinical use leading to the synthesis of the water-soluble analog becatecarin. Early clinical studies with becatecarin were promising with reports from phase I studies noting partial responses in patients diagnosed with gastric cancer [1] or adenocarcinoma of unknown origin [2] and minor responses in patients with gall bladder or pancreatic tumors [1]. In contrast in phase II studies in patients with breast [3] renal cell [4] or colorectal cancer [5] becatecarin displayed relatively modest activity. When becatecarin was combined with cisplatin partial responses were noted in patients with adenocarcinoma of unknown origin non-small cell lung cancer and pancreatic cancer; however progressive disease was eventually reported in all patients [6]. In a phase II study of becatecarin in children with solid tumors only 4 of 126 patients saw clinical benefit and the investigators noted significant myelosupression [7]. Other indolocarbazole topoisomerase inhibitors with structures similar to becatecarin have been developed. NB-506 is a glycosylated derivative of BE-13793C an indolocarbazole topoisomerase I and II inhibitor isolated from the fermentation broth of that served as positive controls for RN-1 2HCl Pgp MRP1 and ABCG2 expression respectively. As seen in Figure 5 A549 Bec150 cells were found to overexpress ABCG2 but not Pgp or MRP1 suggesting that resistance to becatecarin in the A549 Bec150 line is due primarily to ABCG2 expression. Figure 5 ABCG2 expression but not Pgp or MRP1 expression is upregulated in becatecarin-resistant A549 lung carcinoma cells. Microsomal membrane fractions were separated by SDS-PAGE and transferred to a PVDF membrane that was sequentially probed for Pgp MRP1 … DISCUSSION In the present study we demonstrate that becatecarin is a substrate for the ATP binding-cassette half-transporter ABCG2 and that resistance to becatecarin can be reversed by inhibitors of ABCG2 such as FTC. Becatecarin was also found to be fluorescent and FTC-inhibitable becatecarin transport was observed in cell lines by flow cytometry. However becatecarin transport was only observed RN-1 2HCl in drug-resistant cells expressing very high levels of ABCG2 rendering becatecarin a rather insensitive probe for measuring ABCG2 expression. Additionally we describe a becatecarin-resistant cell line RN-1 2HCl derived from A549 lung carcinoma cells A549 Bec150 which was found to overexpress ABCG2 as the primary mechanism of resistance. While becatecarin was found to be transported by ABCG2 it does not appear to be as well transported as other ABCG2 substrates such as mitoxantrone topotecan or SN-38. This is evidenced by the fact that ABCG2-transfected cells are only about 3-fold resistant to becatecarin while they were found to be 15-fold resistant to topotecan. Additionally the becatecarin resistant line was found to be approximately 3-fold resistant to becatecarin but 7-to RN-1 2HCl 15-fold resistant to MX topotecan and SN-38. This is in contrast to studies conducted with the structurally related indolocarbazole compounds compound A (NB-506) [12 29 and J-107088 [12]. Komatani et al generated NB-506-resistant HCT-116 cell lines that were 2400-fold resistant to NB-506 and 230-fold resistant to J-107088 but were only 10-fold resistant to mitoxantrone and 4-fold resistant to topotecan [12]. Similarly Nakagawa et al found that wild-type ABCG2-transfected MCF-7 cells were 298-fold resistant to NB-506 but only 9-fold resistant to mitoxantrone and 7-fold resistant to topotecan. The rebeccamycin derivative becatecarin is also an indolocarbazole but differs from NB-506 and J-107088 by RN-1 2HCl the groups at positions 1 2 6 10 11 and 14 of the indolocarbazole structure [30]. Thus modifications at these positions may affect recognition by the ABCG2 protein but further experiments are needed to confirm this theory. In the present work we demonstrate that continued treatment with becatecarin is able to induce expression of ABCG2 in lung cancer cells despite the fact that becatecarin does not seem to be readily transported by ABCG2. It is possible that becatecarin may induce RN-1 2HCl ABCG2.