Advertisement

Low-intensity ultrasound adjuvant therapy: enhancement of doxorubicin-induced cytotoxicity and the acoustic mechanisms involved

  • Takashi KondoEmail author
  • Toru Yoshida
  • Ryohei Ogawa
  • Mariame A. Hassan
  • Yukihiro Furusawa
  • Qing-Li Zhao
  • Akihiko Watanabe
  • Akihiro Morii
  • Loreto B. FerilJr.
  • Katsuro Tachibana
  • Hiroshi Kitagawa
  • Yoshiaki Tabuchi
  • Ichiro Takasaki
  • Mohammad H. Shehata
  • Nobuki Kudo
  • Kazuhiro Tsukada
Original Article

Abstract

Purpose

In this study, the effects of low-intensity pulsed ultrasound (LIU) as an adjuvant to doxorubicin (DOX) treatment was further investigated in comparison to hyperthermia as another widely used adjuvant. The effects were compared with respect to cell killing and apoptosis induction in U937 cells. Human primary liver cancer (PLC) cells were also used to evaluate the effects of the combinations. The use of an echo contrast agent was investigated for further enhancement of cytotoxicity. Finally, the acoustic mechanisms involved were investigated.

Methods

The effects of different treatment regimens on cell viability were determined using the Trypan blue dye-exclusion test. Apoptosis induction was detected by flow cytometry using fluorescein isothiocyanate-annexin V and propidium iodide staining. The mechanistic study involved electron paramagnetic spin trapping for detecting free radical formation as an indicator of the occurrence of inertial cavitation and spectrophotometry for sucrose hydrolysis as an indicator for noncavitational effects.

Results

The combination treatments exerted synergistic effects on cytotoxicity depending on the acoustic conditions used. The use of LIU as an adjuvant to DOX treatment was shown to be superior to the use of hyperthermia as an adjuvant. Moreover, the combination seems to be promising for other cancer types provided that the acoustic conditions are properly selected with respect to drug concentration. The key ultrasound mechanism responsible for the synergism observed was shown to be the production of free radicals by inertial cavitation. Non-cavitational forces were also shown to contribute to the effect.

Conclusion

This study is motivating to engage in in vivo research with various cancer types as a step toward clinical applicability and is emphasizing on the importance of developing therapeutic protocols for setting LIU parameters with respect to other therapeutic conditions.

Keywords

doxorubicin low-intensity ultrasound apoptosis 

References

  1. 1.
    Wood RW, Loomis AL. The physical and biological effects of high-frequency sound waves of great intensity. Phil Mag S Series 7 1927;7:417.Google Scholar
  2. 2.
    Kremkau FW. Cancer therapy with ultrasound: a historical review. J Clin Ultrasound 1979;7:287–300.PubMedCrossRefGoogle Scholar
  3. 3.
    Blana A, Murat FJ, Walter B, et al. First analysis of the long-term results with transrectal HIFU in patients with localised prostate cancer. Eur Urol 2008;53:1194–1201.PubMedCrossRefGoogle Scholar
  4. 4.
    Chaussy C, Thuroff S, Rebillard X, et al. Technology insight: high-intensity focused ultrasound for urologic cancers. Nat Clin Pract Urol 2005;2:191–198.PubMedCrossRefGoogle Scholar
  5. 5.
    Larkin JO, Casey GD, Tangney M, et al. Effective tumor treatment using optimized ultrasound-mediated delivery of bleomycin. Ultrasound Med Biol 2008;34:406–413.PubMedCrossRefGoogle Scholar
  6. 6.
    Feril LB Jr, Kondo T, Cui ZG, et al. Apoptosis induced by the sonomechanical effects of low-intensity pulsed ultrasound in a human leukemia cell line. Cancer Lett 2005;221:145–152.PubMedCrossRefGoogle Scholar
  7. 7.
    Feng Y, Tian ZM, Wan MX, et al. Low-intensity ultrasoundinduced apoptosis in human gastric carcinoma cells. World J Gastroenterol 2008;14:4873–4879.PubMedCrossRefGoogle Scholar
  8. 8.
    Feril LB Jr, Kondo T. Biological effects of low-intensity ultrasound: the mechanism involved, and its implication on therapy and on biosafety of ultrasound. Rev J Radiat Res 2004;45:479–489.CrossRefGoogle Scholar
  9. 9.
    Yoshida T, Kondo T, Ogawa R, et al. Molecular therapy using ultrasound: mechanisms involved in drug activation, apoptosis induction, gene transfer, and alterations of gene expression. Rev Thermal Med 2007;23:113–122.CrossRefGoogle Scholar
  10. 10.
    Harrison GH, Balcer-Kubiczek EK, Eddy HA. Potentiation of chemotherapy by low-level ultrasound. Int J Radiat Biol 1991;59:1453–1466.PubMedCrossRefGoogle Scholar
  11. 11.
    Loverock P, ter Haar G, Ormerod MG, et al. The effect of ultrasound on the cytotoxicity of adriamycin. Br J Radiol 1990;63:542–546.PubMedCrossRefGoogle Scholar
  12. 12.
    Umemura S, Yumita N, Okano Y, et al. Sonodynamically induced in vitro cell damage enhanced by adriamycin. Cancer Lett 1997;121:195–201.PubMedCrossRefGoogle Scholar
  13. 13.
    Yu T, Bai J, Hu K, et al. The effect of free radical scavenger and antioxidant on the increase in intracellular adriamycin accumulation induced by ultrasound. Ultrason Sonochem 2003;10:33–35.PubMedCrossRefGoogle Scholar
  14. 14.
    Yu T, Wang Z, Jiang S. Potentiation of cytotoxicity of adriamycin on human ovarian carcinoma cell line 3AO by low-level ultrasound. Ultrasonics 2001;39:307–309.PubMedCrossRefGoogle Scholar
  15. 15.
    Yoshida T, Kondo T, Ogawa R, et al. Combination of doxorubicin and low-intensity ultrasound causes a synergistic enhancement in cell killing and an additive enhancement in apoptosis induction in human lymphoma U937 cells. Cancer Chemother Pharmacol 2008;61:559–567.PubMedCrossRefGoogle Scholar
  16. 16.
    Honda H, Kondo T, Zhao QL, et al. Role of intracellular calcium ions and reactive oxygen species in apoptosis induced by ultrasound. Ultrasound Med Biol 2004;30:683–692.PubMedCrossRefGoogle Scholar
  17. 17.
    Honda H, Zhao QL, Kondo T. Effects of dissolved gases and an echo contrast agent on apoptosis induced by ultrasound and its mechanism via the mitochondria-caspase pathway. Ultrasound Med Biol 2002;28:673–682.PubMedCrossRefGoogle Scholar
  18. 18.
    Tabuchi Y, Takasaki I, Zhao QL, et al. Genetic networks responsive to low-intensity pulsed ultrasound in human lymphoma U937 cells. Cancer Lett 2008;270:286–294.PubMedCrossRefGoogle Scholar
  19. 19.
    Ando H, Feril LB Jr, Kondo T, et al. An echo-contrast agent, Levovist, lowers the ultrasound intensity required to induce apoptosis of human leukemia cells. Cancer Lett 2006;242:37–45.PubMedCrossRefGoogle Scholar
  20. 20.
    Correas JM, Kurtisovski E, Bridal SL, et al. Optimizing an ultrasound contrast agent’s stability using in vitro attenuation measurements. Invest Radiol 2002;37:672–679.PubMedCrossRefGoogle Scholar
  21. 21.
    Feril LB Jr, Kondo T. Major factors involved in the inhibition of ultrasound-induced free radical production and cell killing by presonication incubation or by high cell density. Ultrason Sonochem 2005;12:353–357.PubMedCrossRefGoogle Scholar
  22. 22.
    Feril LB Jr, Kondo T, Zhao QL, et al. Enhancement of hyperthermia-induced apoptosis by non-thermal effects of ultrasound. Cancer Lett 2002;178:63–70.PubMedCrossRefGoogle Scholar
  23. 23.
    Hiraoka W, Honda H, Feril LB Jr, et al. Comparison between sonodynamic effect and photodynamic effect with photosensitizers on free radical formation and cell killing. Ultrason Sonochem 2006;13:535–542.PubMedCrossRefGoogle Scholar
  24. 24.
    Woodcock JP. The effect of non-cavitating ultrasound on the hydrolysis of sucrose solutions. Acustica 1969;21:45–50.Google Scholar
  25. 25.
    Feril LB Jr, Kondo T, Zhao QL, et al. Enhancement of ultrasoundinduced apoptosis and cell lysis by echo-contrast agents. Ultrasound Med Biol 2003;29:331–337.PubMedCrossRefGoogle Scholar
  26. 26.
    Ogawa R, Kondo T, Honda H, et al. Effects of dissolved gases and an echo contrast agent on ultrasound-mediated in vitro gene transfection. Ultrason Sonochem 2002;9:197–203.PubMedCrossRefGoogle Scholar
  27. 27.
    Watanabe A, Otake R, Nozaki T, et al. Effects of microbubbles on ultrasound-mediated gene transfer in human prostate cancer PC3 cells: comparison among Levovist, YM454, and MRX-815H. Cancer Lett 2008;265:107–112.PubMedCrossRefGoogle Scholar
  28. 28.
    Duvshani-Eshet M, Baruch L, Kesselman E, et al. Therapeutic ultrasound-mediated DNA to cell and nucleus: bioeffects revealed by confocal and atomic force microscopy. Gene Ther 2006;13:163–172.PubMedCrossRefGoogle Scholar
  29. 29.
    Sauer H, Putz V, Fischer K, et al. Increased doxorubicin uptake and toxicity in multicellular tumour spheroids treated with DC electrical fields. Br J Cancer 1999;80:1204–1213.PubMedCrossRefGoogle Scholar
  30. 30.
    Keizer HG, Pinedo HM, Schuurhuis GJ, et al. Doxorubicin (adriamycin): a critical review of free radical-dependent mechanisms of cytotoxicity. Pharmacol Ther 1990;47:219–231.PubMedCrossRefGoogle Scholar

Copyright information

© The Japan Society of Ultrasonics in Medicine 2009

Authors and Affiliations

  • Takashi Kondo
    • 1
    Email author
  • Toru Yoshida
    • 2
  • Ryohei Ogawa
    • 1
  • Mariame A. Hassan
    • 1
  • Yukihiro Furusawa
    • 1
  • Qing-Li Zhao
    • 1
  • Akihiko Watanabe
    • 3
  • Akihiro Morii
    • 3
  • Loreto B. FerilJr.
    • 4
  • Katsuro Tachibana
    • 4
  • Hiroshi Kitagawa
    • 5
  • Yoshiaki Tabuchi
    • 6
  • Ichiro Takasaki
    • 6
  • Mohammad H. Shehata
    • 7
  • Nobuki Kudo
    • 8
  • Kazuhiro Tsukada
    • 2
  1. 1.Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical SciencesUniversity of ToyamaToyamaJapan
  2. 2.Second Department of Surgery, Graduate School of Medicine and Pharmaceutical SciencesUniversity of ToyamaToyamaJapan
  3. 3.Department of Urology, Graduate School of Medicine and Pharmaceutical SciencesUniversity of ToyamaToyamaJapan
  4. 4.Department of AnatomyFukuoka University School of MedicineFukuokaJapan
  5. 5.Laboratory of Animal Histophysiology, Division of Animal Science, Department of Bioresource Science, Graduate School of Agricultural ScienceKobe UniversityKobeJapan
  6. 6.Life Science Research CenterUniversity of ToyamaToyamaJapan
  7. 7.Department of Biochemistry, Graduate School of Medicine and Pharmaceutical SciencesUniversity of ToyamaToyamaJapan
  8. 8.Laboratory of Biomedical Instrumentation and Measurements, Graduate School of Information Science and TechnologyHokkaido UniversitySapporoJapan

Personalised recommendations