ABCG2 Inhibitor YHO-13351 Sensitizes Cancer Stem/ Initiating-like Side Population Cells to Irinotecan

Background/Aim: The aim of this study was to determine the efficacy of the combination of irinotecan and newly-synthesized ABCG2 (breast cancer-resistant protein) inhibitor YHO-13351 in cancer chemotherapy. Materials and Methods: Side population (SP) and non-SP cells from the human cervical carcinoma cell line HeLa were isolated by fluorescence-activated cell sorting. The antitumor activity of combination therapy with irinotecan and YHO-13351 was evaluated in xenograft studies in athymic BALB/c nude mice. Results: While SP cells exhibited cancer stem/initiating cell- like properties and low sensitivity to irinotecan-alone, YHO- 13351 sensitized them to irinotecan in both in vitro and in vivo studies. YHO-13351 in conjunction with irinotecan reduced the increase of the SP cell ratio in the tumors compared to those observed with treatment with irinotecan- alone. Conclusion: Combination therapy with irinotecan and YHO-13351 would not only accelerate the antitumor effect of this regimen, but also play a crucial role in preventing resistance or relapse. Two major problems encountered in the treatment of cancer are resistance to chemotherapy and recurrence. One common approach to overcoming resistance is to try new combinations of anticancer agents with different modes of action (1, 2). The results, however, are not always satisfactory, even though benefits may be observed in some cases. Therefore, there is an urgent need to develop more effective means of sensitizing tumors to conventional chemotherapeutics and to completely eradicate cancerous cells. One cause of resistance is an ATP-binding cassette (ABC) transporter-induced decrease in the intracellular accumulation of a drug (3). Therefore, much effort has been devoted to developing ABC transporter inhibitors such as ABCB1 (P- glycoprotein) and ABCG2 (breast cancer-resistant protein) in hope that this would bring about a breakthrough in the amelioration of drug resistance (4-6). ABCG2, in particular, is involved in the efflux of several anticancer agents, including irinotecan and its active metabolite, SN-38 (7, 8), one of the most widely prescribed drugs for many types of cancer (9). In addition, ABCG2 is a key molecule in the characterization of side population (SP) cells in the hematopoietic system, which are identified by their ability to exclude the fluorescent vital dye Hoechst 33342 (10). The ABCG2-positive subset of tumor cells is also frequently identified by its ability to exclude Hoechst, and it is often enriched with cells with cancer stem/initiating-like phenotypes (11-13). Some studies have attributed failure of chemotherapy to the survival of multi-drug chemoresistant cancer stem/initiating cells (14-16). YHO-13177, (Z)-2-(3,4-dimethoxyphenyl)-3-(5-(4- hydroxypiperidin-1-yl) thiophen-2-yl) acrylonitrile, is a novel acrylonitrile derivative discovered by screening for ABCG2 inhibitors (17). YHO-13351, diethylaminoacetic acid 1-{5- ((Z)-2-cyano-2-(3,4-dimethoxyphenyl)vinyl)thiophen-2- yl}piperidin-4-yl ester methanesulfonate, is a water-soluble pro-drug of YHO-13177. Co-administration of irinotecan with YHO-13351 significantly increased survival time in mice inoculated with ABCG2-transduced murine leukemia P388 cells and suppressed tumor growth in a ABCG2- transduced HTC116 human colonic cancer xenograft model, whereas irinotecan-alone had little effect in these tumor models (17). Thus, the combination of YHO-13351 and irinotecan offers a potentially promising chemotherapy regimen capable of completely eradicating tumors by targeting cancer stem/initiating cells. In this study, SP cells isolated from human cervical carcinoma HeLa cells were characterized by using hallmarks of stemness. The sensitivity of HeLa SP and non-SP cells to irinotecan (SN-38) was evaluated. The effect of YHO-13351 (YHO-13177) on this sensitivity was examined in in vitro cell culture and an in vivo xenograft mouse model. The superiority of combination cancer chemotherapy with irinotecan and YHO-13351 was also investigated.