J Cancer 2016; 7(11):1524-1533. doi:10.7150/jca.15181

Research Paper

Inhibition of Regulatory Volume Decrease Enhances the Cytocidal Effect of Hypotonic Shock in Hepatocellular Carcinoma

Michihiro Kudou1*, Atsushi Shiozaki1✉*, Toshiyuki Kosuga1*, Daisuke Ichikawa1, Hirotaka Konishi1, Ryo Morimura1, Shuhei Komatsu1, Hisashi Ikoma1, Hitoshi Fujiwara1, Kazuma Okamoto1, Shigekuni Hosogi2, Takashi Nakahari2, Yoshinori Marunaka2, 3, Eigo Otsuji1

1. Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto, 602-8566, Japan;
2. Departments of Molecular Cell Physiology and Bio-Ionomics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, 602-8566, Japan;
3. Japan Institute for Food Education and Health, Heian Jogakuin (St. Agnes') University, Kyoto, 602-8013, Japan.
* These authors contributed equally to this work.

Abstract

Background: Hypotonic shock induces cytocidal effects through cell rupture, and cancer therapy based on this mechanism has been clinically administered to hepatocellular carcinoma patients. We herein investigated the effectiveness of hypotonic shock combined with the inhibition of regulatory volume decrease as cancer therapy for hepatocellular carcinoma.

Methods: Morphological changes in human hepatocellular carcinoma cell lines were observed under a differential interference contrast microscope connected to a high-speed digital video camera. Cell volume changes under hypotonic shock with or without chloride, potassium, or water channel blockers were observed using a high-resolution flow cytometer. In order to investigate cytocidal effects, the number of surviving cells was compared after exposure to hypotonic solution with and without each channel blocker (re-incubation experiment).

Results: Video recordings showed that cells exposed to distilled water rapidly swelled and then ruptured. Cell volume measurements revealed regulatory volume decrease under mild hypotonic shock, whereas severe hypotonic shock increased the number of broken fragments as a result of cell rupture. Moreover, regulatory volume decrease was inhibited in cells treated with each channel blocker. Re-incubation experiments showed the cytocidal effects of hypotonic shock in cells exposed to hypotonic solution, and additional treatments with each channel blocker enhanced these effects.

Conclusion: The inhibition of regulatory volume decrease with chloride, potassium, or water channel blockers may enhance the cytocidal effects of hypotonic shock in hepatocellular carcinoma. Hypotonic shock combined with the inhibition of regulatory volume decrease was a more effective therapy than hypotonic shock alone.

Keywords: Hepatocellular carcinoma, Regulatory volume decrease, hypotonic shock, potassium ion transport, chloride ion transport.

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How to cite this article:
Kudou M, Shiozaki A, Kosuga T, Ichikawa D, Konishi H, Morimura R, Komatsu S, Ikoma H, Fujiwara H, Okamoto K, Hosogi S, Nakahari T, Marunaka Y, Otsuji E. Inhibition of Regulatory Volume Decrease Enhances the Cytocidal Effect of Hypotonic Shock in Hepatocellular Carcinoma. J Cancer 2016; 7(11):1524-1533. doi:10.7150/jca.15181. Available from http://www.jcancer.org/v07p1524.htm