Previous studies highlighted that aminopeptidase N (APN)/CD13 acts as a scavenger in the survival of hepatocellular carcinoma (HCC) stem cells by reducing reactive oxygen species (ROS) levels. Hence, it has been proposed that APN/CD13 inhibition can increase cellular ROS levels and sensitize cells to chemotherapeutic agents. Although ubenimex, also known as bestatin, competitively inhibits proteases such as APN/CD13 on the cellular membrane and it is clinically used for patients with acute myeloid leukemia and lymphedema, research has demonstrated that higher concentrations of the agent induce the death of APN/CD13+ HCC stem cells. In this study, we developed a poly(ethylene glycol)–poly(lysine) block copolymer–ubenimex conjugate (PEG-b-PLys(Ube)) to increase the efficacy of reagents in APN/CD13+ cancer stem cells. Exposure to PEG-b-PLys(Ube) increased the intracellular ROS concentration by inhibiting APN enzyme activity, permitting the induction of apoptosis and attenuation of HCC cell proliferation. In addition, PEG-b-PLys(Ube) exhibited a relatively stronger antitumor effect in mice than PEG-b-PLys alone or phosphate-buffered saline. Moreover, an isobologram analysis revealed that combinations of fluorouracil, cisplatin, or doxorubicin with PEG-b-PLys(Ube) exhibited synergistic effects. This study demonstrated that PEG-b-PLys(Ube) does not impair the properties of ubenimex and exerts a potent antitumor effect.
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We thank M. Ozaki and Y. Noguchi for technical assistance and the laboratory staff for their helpful discussions.
This work received financial support from grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (grant no. 17H04282; 17K19698; 16K15615; 15H05791; P-DIRECT; P-CREATE; AMED−Japan Cancer Research Project).
Conception and design: RT, MK, HT, NN, HI. Development of methodology: RT, MK, NN, HI. Acquisition of the data (e.g., provided animals, acquired and managed patients, provided facilities): RT, MK, HT, TN, AA, JK, YU, KM, KA, TO, NN, HI. Analysis and interpretation of the data (e.g., statistical analysis, biostatistics, computational analysis): RT, MK, NH, YI, DY, DS, TA, TK, KK, KG, SK, TS, YD, MM, HI. Writing, review, and/or revision of the manuscript: RT, MK, HE, HT, TN, NH, HI. Study supervision: MM, HI.
Conflict of interest
YD, MM, HI received Institutional endowments were received from Taiho Pharmaceutical Co., Ltd., Unitech Co., Ltd. (Chiba, Japan), IDEA Consultants, Inc. (Tokyo, Japan), and Kinshu-kai Medical Corporation (Osaka, Japan); DS, TK, TS, YD, MM received Chugai Co., Ltd., Yakult Honsha Co., Ltd., and Merck & Co., Ltd (YD, MM, TS). The remaining authors declare that they have no conflict of interest.
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Toshiyama, R., Konno, M., Eguchi, H. et al. Poly(ethylene glycol)–poly(lysine) block copolymer–ubenimex conjugate targets aminopeptidase N and exerts an antitumor effect in hepatocellular carcinoma stem cells. Oncogene 38, 244–260 (2019). https://doi.org/10.1038/s41388-018-0406-x
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