Apatinib (YN968D1) reverses multidrug resistance by inhibiting the function of multiple ATP-binding cassette transporters Yanjun Mi, Liwu Fu State Key Laboratory of oncology in southern China, Cancer Center, Sun Yat-sen University, Guangzhou, 510060, China Email: Fulw@mail.sysu.edu.cn

Background A successful cancer chemotherapy is limited by multidrug resistance (MDR). The major inducers of MDR are the ABC transporters, such as ABCB1 (anthracyclins, vinca alkaloids, taxanes, and epipodophyllotoxins), ABCG2 (mitoxantrone, anthracyclins, topoisomerase I inhbitors, methotrexate). Combination of modulator with conventional chemotherapeutic drugs to restore the sensitivity of MDR cells is a main strategy for overcoming MDR.

Background Tyrosine kinase inhibitors (TKIs) belong to a new class of anticancer drugs. TKIs including lapatinib, erlotinib and sunitinib, have been shown to overcome ABC transporters-mediated MDR in cancer cells. Apatinib (YN968D1) is an oral small-molecule TKI that targets VEGFR-2, RET, c-Kit and c-Src tyrosine kinases. It is conceivable that apatinib may inhibit functions of ABC transporters by binding to their ATP-binding sites.

Cytotoxicity of apatinib alone in all experimental cell lines ABCB1 ABCG2 More than 85% of cells were viable at concentrations of apatinib up to 3.0 μM in all experimental cells.

Apatinib reversed MDR in vitro Compounds IC50 ± SD (μM) (fold-reversal) KB KBv200 (ABCB1) Doxorubicin 0.029 ± 0.002 (1.00) 2.277 ± 0.134 + 0.75 μM Apatinib 1.259 ± 0.099** (1.81) + 1.5 μM Apatinib 0.030 ± 0.001 (0.98) 0.964 ± 0.070** (2.36) + 3.0 μM Apatinib 0.028 ± 0.003 (1.04) 0.337 ± 0.016** (6.81) + 10 μM Verapamil 0.029 ± 0.003 0.106 ± 0.006** (21.4) Paclitaxel 0.0018 ± 0.0002 0.296 ± 0.027 0.0018 ± 0.0003 0.142 ± 0.013** (2.08) 0.0019 ± 0.0002 (0.95) 0.045 ± 0.004** (6.58) 0.009 ± 0.001** (32.9) 0.007 ± 0.001** (42.3) Cispatin 0.726 ± 0.055 1.284 ± 0.141 0.714 ± 0.057 (1.01) 1.292 ± 0.125 (0.99) MCF-7 MCF-7/adr (ABCB1) 0.344 ± 0.037 (1.00 ) 11.504 ± 1.186 0.349 ± 0.011 3.021 ± 0.196** (3.81) 0.331 ± 0.019 2.177 ± 0.273** (5.28) 0.350 ± 0.036 (0.98 ) 0.854 ± 0.056** (13.5) 0.340 ± 0.038 0.540 ± 0.076** (21.3) 5.811 ± 0.533 4.622 ± 0.371 5.624 ± 0.211 (1.03 ) 4.531 ± 0.352 (1.02)

Apatinib reversed MDR in vitro Compounds IC50 ± SD (μM) (fold-reversal) S1 S1-M1-80 (ABCG2) Mitoxantrone 0.194 ± 0.027 (1.00) 13.651 ± 0.922 + 0.75 μM Apatinib 0.196 ± 0.041 (0.98) 6.434 ± 0.478** (2.12) + 1.5 μM Apatinib 0.185 ± 0.058 (1.05) 2.070 ± 0.621** (6.59) + 3.0 μM Apatinib 0.136 ± 0.067 (1.42) 1.188 ± 0. 495** (11.5) + 2.5 μM FTC 0.188 ± 0.011 (1.03) 0.892 ± 0.056** (15.3) Topotecan 0.262 ± 0.042 10.281 ± 0.455 0.264 ± 0.022 (0.99) 4.089 ± 0.026** (2.51) 0.247 ± 0.017 2.037 ± 0.083** (5.04) 0.196 ± 0.055 (1.33) 1.188 ± 0.055** (8.65) 0.254 ± 0.016 (1.02) 0.745 ± 0.068** (13.8) Cisplatin 12.811 ± 1.181 12.092 ± 1.322 12.280 ± 1.990 (1.04) 12.143 ± 1.452 HL60 HL60/ADR(ABCC1) Doxorubicin 0.036 ± 0.002 5.704 ± 0.378 0.035 ± 0.002 5.493 ± 0.289 0.037 ± 0.002 (0.97) 5.528 ± 0.515 0.033 ± 0.004 (1.09) 5.719 ± 0.595

Apatinib reversed MDR in vitro Compounds IC50 ± SD (μM) (fold-reversal)   HEK293/pcDNA3.1 HEK293/ABCG2-R2 HEK293/ABCB1 Mitoxantrone 0.0569 ± 0.0035 (1.00) 1.3460 ± 0.3143 0.1381 ± 0.0274 + 3 μM Apatinib 0.0349 ± 0.0097 (1.63) 0.0655 ± 0.0199** (20.55) 0.0616 ± 0.0357** (2.24) + 3 μM FTC 0.0528 ± 0.0093 (0.98) 0.0687 ± 0.0126** (19.59) - + 3 μM PSC833 0.0543 ± 0.0069 (1.04) 0.0688 ± 0.0459** (2.01) SN-38 0.0073 ± 0.0003 0.1530 ± 0.1636 0.0045 ± 0.0009 (1.62) 0.0079 ± 0.0021** (19.37) 0.0050 ± 0.0003 (1.46) 0.0085 ± 0.0016** (18.00) Vincristine 0.0437 ± 0.0022 (1.00 0.6405 ± 0.0349 0.0335 ± 0.0039 (1.30) 0.1792 ± 0.0485** (3.57) + 10 μM Verapamil 0.0450 ± 0.0003 (0.97) 0.0514 ± 0.0025** (12.46) Doxorubicin 0.0724 ± 0.0054 1.0821 ± 0.5424 0.0512 ± 0.0033 (1.41) 0.3168 ± 0.0045** (3.42) 0.0957 ± 0.0142 (0.77) 0.0964 ± 0.0153** (11.23) Cisplatin 1.8240 ± 0.4728 1.6521 ± 0.3892 1.4899 ± 0.5321 1.5256 ± 0.3717 (1.19) 1.4193 ± 0.4820 (1.16) 1.6479 ± 0.2402 (0.90)

Potentiation of antitumor effects of paclitaxel by apatinib in a xenograft model of KBv200 cells in nude mice Control: saline Apatinib: 70 mg/Kg, P.O., q3dX4 Paclitaxel:18 mg/kg, i.p., q3dx4 Combination: 70 mg/Kg, P.O., q3dx4+18 mg/kg, i.p., q3dx4 The combination of Apatinib and paclitaxel produced a significantly inhibitory effect on the growth of the xenografts but did not result in the increased toxicity 52%

Effect of apatinib on the accumulation of doxorubicin

Effect of apatinib on the accumulation of rhodamine 123

Apatinib ABCB1 and ABCG2 ATPase activity Expression of ABCB1 and ABCG2 Intracellular accumulation of drugs in the MDR cells Reverse ABCB1- and ABCG2-mediated MDR

Effect of apatinib on the expression of ABCB1 and ABCG2

Effect of apatinib on the expression of ABCB1 and ABCG2

Effect of apatinib on ABCB1 and ABCG2 ATPase activity 950nmol/L 10-12nmol/L

Effect of apatinib on photoaffinity labeling of ABCB1 and ABCG2 with [125I]-IAAP IC50 = 2.9 ± 0.40 (μmol/L) IC50 = 11 ± 4 (nmol/L)

Effect of apatinib on the phosphorylation of AKT and ERK1/2

Apatinib MDR ABCB1 and ABCG2 ATPase activity Expression of ABCB1 and ABCG2 Intracellular accumulation of drugs in the MDR cells Blockade of AKT and ERK1/2 activation Reverse MDR

Conclusion Apatinib reverses ABCB1- and ABCG2-mediated MDR in vitro and in vivo by directly inhibiting ABCB1 and ABCG2 function, resulting in elevated intracellular concentrations of substrate chemotherapeutic drugs. The reversal of MDR is not associated with the blockade of tyrosine kinases. These findings may be useful for cancer combinational therapy of chemotherapeutic drug with apatinib in the clinic.

Acknowledgments We like to thank Professor Suresh V. Ambudkar (Center for Cancer Research, NCI, NIH, Bethesda) and Zhe-Sheng Chen (College of Pharmacy and Allied Health Professions, St. John’s University ) for collaborating to finish this work.

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