Abstract

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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Acknowledgements

We thank M. Ozaki and Y. Noguchi for technical assistance and the laboratory staff for their helpful discussions.

Funding

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).

Author contributions

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.

Author information

Author notes

  1. These authors contributed equally: Reishi Toshiyama, Masamitsu Konno

Affiliations

  1. Departments of Gastroenterological Surgery, Graduate School of Medicine, Osaka University, Suita, Osaka, 565-0871, Japan

    • Reishi Toshiyama
    • , Hidetoshi Eguchi
    • , Takehiro Noda
    • , Naotsugu Haraguchi
    • , Yuji Ueda
    • , Katsunori Matsushita
    • , Kei Asukai
    • , Tomofumi Ohashi
    • , Yoshifumi Iwagami
    • , Daisaku Yamada
    • , Tadafumi Asaoka
    • , Koichi Kawamoto
    • , Kunihito Gotoh
    • , Shogo Kobayashi
    • , Yuichiro Doki
    •  & Masaki Mori
  2. Departments of Frontier Science for Cancer and Chemotherapy, Graduate School of Medicine, Osaka University, Suita, Osaka, 565-0871, Japan

    • Reishi Toshiyama
    • , Masamitsu Konno
    • , Ayumu Asai
    • , Yuji Ueda
    • , Katsunori Matsushita
    • , Kei Asukai
    • , Tomofumi Ohashi
    • , Daisuke Sakai
    • , Toshihiro Kudo
    • , Koichi Kawamoto
    •  & Taroh Satoh
  3. Departments of Medical Data Science, Graduate School of Medicine, Osaka University, Suita, Osaka, 565-0871, Japan

    • Reishi Toshiyama
    • , Ayumu Asai
    • , Jun Koseki
    • , Yuji Ueda
    • , Katsunori Matsushita
    • , Kei Asukai
    • , Tomofumi Ohashi
    •  & Hideshi Ishii
  4. Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa, Japan

    • Hiroyasu Takemoto
    •  & Nobuhiro Nishiyama

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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.

Corresponding authors

Correspondence to Masaki Mori or Hideshi Ishii.

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DOI

https://doi.org/10.1038/s41388-018-0406-x