Ethyl 2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (CXL017): a novel scaffold that resensitizes multidrug resistant leukemia cells to chemotherapy

Abstract

Multidrug resistance (MDR) is a major hurdle in the treatment of cancer, and there is a pressing need for new therapies. We have recently developed ethyl 2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (CXL017), derived from a dual inhibitor of Bcl-2 and SERCA proteins, sHA 14-1, with selective cytotoxicity toward MDR cancer cell lines in vitro. In this study, we present new evidence for its therapeutic potential in treatment of MDR cancers and offer mechanistic insights toward its preferential targeting of drug-resistant cancer. CXL017 selectively suppressed the growth of tumors derived from the MDR cancer cell line, HL60/MX2, in vivo. In addition, even after chronic exposure to CXL017, HL60/MX2 failed to develop stable resistance to CXL017, whereas it acquired >2000-fold resistance to cytarabine (Ara-C), the major first-line chemotherapy for the treatment of acute myeloid leukemia (AML). Remarkably, instead of acquiring further cross-resistance, HL60/MX2 cells exposed to CXL017 were resensitized to standard therapies (10- to 100-fold). Western blotting analyses revealed that CXL017 exposure significantly down-regulated Mcl-1 and Bax and up-regulated Noxa, Bim, Bcl-X(L), SERCA2, and SERCA3 proteins, along with a reduction in endoplasmic reticulum (ER) calcium content. Given the well-established functions of Bcl-2 family proteins and ER calcium in drug resistance, our results suggest that the down-regulation of Mcl-1 and the up-regulation of Noxa and Bim along with the decrease in ER calcium content are likely responsible for CXL017-induced resensitization of MDR cancer cells. These data also demonstrate the unique potential of CXL017 to overcome MDR in cancer treatment.

Figure 1

The chemical structures of sHA 14-1 and CXL017.

Figure 2. Effect of CXL017 (100mg/kg/day i.p.) on the growth of HL60 and HL60/MX2 inoculated tumors in athymic nude mice

Each mouse was inoculated subcutaneously with HL60/MX2 (right flank) and HL60 cells (left flank). HL60/MX2 inoculated tumors were allowed to grow to the size of 40mm³ and mice were randomized into two groups. Control mice received vehicle whereas treated mice received 100mg/kg/day of CXL017 for three weeks, 6 days a week. A, Bodyweight record of mice. Points, average body weight for each group (n=10); bars, SEM. B, Tumor size record induced by HL60. Points, average tumor volumes for each group(n=10); bars, SEM. P > 0.05 for the comparison between treated and control groups. C, Tumor size record induced by HL60/MX2. Points, average tumor volumes for each group (n=10); bars, SEM. *, P < 0.05 for the comparison between treated and control groups.

Figure 2. Effect of CXL017 (100mg/kg/day i.p.) on the growth of HL60 and HL60/MX2 inoculated tumors in athymic nude mice

Each mouse was inoculated subcutaneously with HL60/MX2 (right flank) and HL60 cells (left flank). HL60/MX2 inoculated tumors were allowed to grow to the size of 40mm³ and mice were randomized into two groups. Control mice received vehicle whereas treated mice received 100mg/kg/day of CXL017 for three weeks, 6 days a week. A, Bodyweight record of mice. Points, average body weight for each group (n=10); bars, SEM. B, Tumor size record induced by HL60. Points, average tumor volumes for each group(n=10); bars, SEM. P > 0.05 for the comparison between treated and control groups. C, Tumor size record induced by HL60/MX2. Points, average tumor volumes for each group (n=10); bars, SEM. *, P < 0.05 for the comparison between treated and control groups.

Figure 3. Characterization of HL60 and HL60/MX2 cell lines

A, HL60 and HL60/MX2 cells were treated with PE conjugated anti-P-gp for 1 h. The cells were then washed and fixed and analyzed using FACS caliber. Three independent experiments were averaged and the figure depicts the fold difference of P-gp expression between HL60 and HL60/MX2. B, HL60 and HL60/MX2 cells were treated with PE conjugated anti-BCRP for 1 h. The cells were then washed and fixed and analyzed using FACS caliber. Three independent experiments were averaged and the figure depicts the fold difference of BCRP expression between HL60 and HL60/MX2. C, Total cell lysates from HL60 and HL60/MX2 were separated by SDS-PAGE and immunoblotted with mouse monoclonal anti-SERCA2, SERCA3, Bax, Noxa or rabbit polyclonal anti-Bak and Bim. Mouse monoclonal anti-β-actin was used as a protein loading control and the β-actin blot shown herein was from the SERCA3 experiment as a representative. Three independent experiments were conducted with similar results. D, ER calcium content in HL60/MX2 cells and that in HL60. ER calcium levels were measured in HL60/MX2 and HL60 cells as described in Materials and Methods. Three independent experiments were averaged with SEM shown.

Figure 4. Long-term exposure of CXL017 and Ara-C to HL60 and HL60/MX2 cells

Drug resistance of HL60 and HL60/MX2 cells was developed as described in Material and Methods. Briefly, HL60 and HL60/MX2 cells were treated with increasing concentrations of CXL017 and Ara-C respectively for 6 months and their sensitivity to drugs was evaluated by using the CellTiter-blue assay. For drug sensitivity, three independent experiments were conducted with similar results. A, Dose-dependent effect of CXL017 on the cell viability of HL60/MX2 and HL60/MX2/CXL017-6 cells. Points, mean (n = 3); bars, SEM. B, Dose-dependent effect of CXL017 on the cell viability of HL60 and HL60/CXL017-6 cells. Points, mean (n = 3); bars, SEM. C, Dose-dependent effect of Ara-C on the cell viability of HL60/MX2 and HL60/MX2/Ara-C-6 cells. Points, mean (n = 3); bars, SEM. D, Dose-dependent effect of Ara-C on the cell viability of HL60 and HL60/Ara-C-6 cells. Points, mean (n = 3); bars, SEM.

Figure 5. Characterization of molecular changes in CXL017 or Ara-C treated HL60/MX2 Cells

A, Total cell lysates from HL60/MX2/Ara-C, HL60/MX2, HL60/MX2/CXL017-6, and HL60/MX2/CXL017 were separated by SDS-PAGE and immunoblotted with mouse monoclonal anti-SERCA2, SERCA3, Bcl-2, Bax, Noxa or rabbit polyclonal anti-Mcl-1, Bcl-X_L, Bak, or Bim. Mouse monoclonal anti-β-actin was used as a protein loading control and the β-actin blot shown herein was from the Mcl-1 experiment as a representative. Three independent experiments were conducted with similar results. B, ER calcium levels were measured in HL60/MX2, HL60/MX2/CXL017, and HL60/MX2/Ara-C cells as described in Materials and Methods. Three independent experiments were averaged and the ratio of 340/380 nm was plotted with SEM shown.