Cancer Chemotherapy and Pharmacology

, Volume 61, Issue 4, pp 559–567 | Cite as

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

  • Toru Yoshida
  • Takashi Kondo
  • Ryohei Ogawa
  • Loreto B. FerilJr
  • Qing-Li Zhao
  • Akihiko Watanabe
  • Kazuhiro Tsukada
Original Article



Potential clinical use of ultrasound (US) in enhancing the effects of anticancer drugs in the treatment of cancers has been highlighted in previous reports. Increased uptake of drugs by the cancer cells due to US has been suggested as a mechanism. However, the precise mechanism of the enhancement has not yet been elucidated. Here, the combined effects of low-intensity pulsed US and doxorubicin (DOX) on cell killing and apoptosis induction of U937 cells, and mechanisms involved were investigated.


Human myelomonocytic lymphoma U937 cells were used for the experiments. Experiments were conducted in 4 groups: (1) non-treated, (2) DOX treated (DOX), (3) US treated (US), and (4) combined (DOX + US). In DOX +US, cells were exposed to 5 μM DOX for 30 min and sonicated by 1 MHz pulsed US (PRF 100 Hz, DF 10%) at intensities of 0.2–0.5 W/cm2 for 60 s. The cells were washed and incubated for 6 h. The viability was evaluated by Trypan blue dye exclusion test and apoptosis and incorporation of DOX was assessed by flow cytometry. Involvement of sonoporation in molecular incorporation was evaluated using FITC-dextran, hydroxyl radical formation was measured by electron paramagnetic resonance-spin trapping, membrane alteration including lipid peroxidation and membrane fluidity by DOX was evaluated using cis-parinaric acid and perylene fluorescence polarization method, respectively.


Synergistic enhancement in cell killing and additive enhancement in induction of apoptosis were observed at and above 0.3 W/cm2. No enhancement was observed at 0.2 W/cm2 in cell killing and induction of apoptosis. Hydroxyl radicals formation was detected at and above 0.3 W/cm2. The radicals were produced more in the DOX + US than US alone. Incorporation of DOX was increased 13% in DOX + US (vs. DOX) at 0.5 W/cm2. Involvement of sonoporation for increase of drug uptake was suggested by experiment using FITC-labeled dextran. We made the hypothesis that DOX treatment made the cells weaken against the mechanical effect of the US. Although treatment of DOX at 5 μM for 30 min did not affect lipid peroxidation and fluidity of cell membrane significantly, higher concentration and longer treatment of DOX induced the significant alteration of cell membrane.


Mechanisms of enhancements could be (1) increase in incorporation of the DOX by US involved with sonoporation, (2) enhancement of the cavitation by DOX. Cavitation is required for the enhancement of the effect of DOX. Although the precise involvement of the membrane modifications by DOX in the enhancement remains to be elucidated, they could be involved in the latent effects.


Doxorubicin Low-intensity ultrasound Apoptosis 



This study was in a part supported by the Research and Development Committee Program of the Japan Society of Ultrasonics in Medicine.


  1. 1.
    Awato S, Kinoshita K, Ikegami A (1977) Dynamic structure of lipid bilayers studied by nanosecond fluorescence techniques. Biochemistry 16:2319–2324CrossRefGoogle Scholar
  2. 2.
    Bachur NR, Gordon SL, Gee MV (1977) Anthracycline antibiotic augmentation of microsomal electron transport and free radical formation. Mol Pharmacol 13:901–910PubMedGoogle Scholar
  3. 3.
    Bachur NR, Yu F, Johnson R, Hickey R, Wu Y, Malkas L (1992) Helicase inhibition by anthracycline anticancer agents. Mol Pharmacol 41:993–998PubMedGoogle Scholar
  4. 4.
    Bao S, Thrall BD, Miller DL (1997) Transfection of a reporter plasmid into cultured cells by sonoporation in vitro. Ultrasound Med Biol 23:953–959PubMedCrossRefGoogle Scholar
  5. 5.
    Benchekroun MN, Robert J (1992) Measurement of doxorubicin-induced lipid peroxidation under the conditions that determine cytotoxicity in cultured tumor cells. Anal Biochem 201:326–330PubMedCrossRefGoogle Scholar
  6. 6.
    Capranico G, Kohn KW, Pommier Y (1990) Local sequence requirements for DNA cleavage by mammalian topoisomerase II in the presence of doxorubicin. Nucleic Acids Res 25:6611–6619CrossRefGoogle Scholar
  7. 7.
    Fechheimer M, Boylan JF, Parker S, Sisken JE, Patel GL, Zimmer SG (1987) Transfection of mammalian cells with plasmid DNA by scrape loading and sonication loading. Proc Natl Acad Sci USA 84:8463–8467PubMedCrossRefGoogle Scholar
  8. 8.
    Feril LB Jr, Kondo T (2005) Major factors involved in the inhibition of ultrasound-induced free radical production and cell killing by pre-sonication incubation or by high cell density. Ultrason Sonochem 12:353–357PubMedCrossRefGoogle Scholar
  9. 9.
    Feril LB Jr, Kondo T, Cui ZG, Tabuchi Y, Zhao QL, Ando H, Misaki T, Yoshikawa H, Umemura S (2005) Apoptosis induced by the sonomechanical effects of low intensity pulsed ultrasound in a human leukemia cell line. Cancer Lett 221:145–152PubMedCrossRefGoogle Scholar
  10. 10.
    Feril LB Jr, Kondo T, Zhao QL, Ogawa R (2002) Enhancement of hyperthermia-induced apoptosis by non-thermal effects of ultrasound. Cancer Lett 178:63–70PubMedCrossRefGoogle Scholar
  11. 11.
    Feril LB Jr, Kondo T, Zhao QL, Ogawa R, Tachibana K, Kudo N, Fujimoto S, Nakamura S (2003) Enhancement of ultrasound-induced apoptosis and cell lysis by echo-contrast agents. Ultrasound Med Biol 29:331–337PubMedCrossRefGoogle Scholar
  12. 12.
    Feril LB, Kondo T, Umemura S, Tachibana K, Manalo AH, Riesz P (2002) Sound waves and antineoplastic drugs: the possibility of an enhanced combined anticancer therapy. J Med Ultrasonics 29Google Scholar
  13. 13.
    Harrison GH, Balcer-Kubiczek EK, Eddy HA (1991) Potentiation of chemotherapy by low-level ultrasound. Int J Radiat Biol 59:1453–1466PubMedCrossRefGoogle Scholar
  14. 14.
    Harrison GH, Balcer-Kubiczek EK, Gutierrez PL (1996) In vitro action of continuous-wave ultrasound combined with adriamycin, X rays or hyperthermia. Radiat Res 145:98–101PubMedCrossRefGoogle Scholar
  15. 15.
    Harrison GH, Balcer-Kubiczek EK, Gutierrez PL (1996) In vitro mechanisms of chemopotentiation by tone-burst ultrasound. Ultrasound Med Biol 22:355–362PubMedCrossRefGoogle Scholar
  16. 16.
    Hedley D, Chow S (1992) Flow cytometric measurement of lipid peroxidation in vital cells using parinaric acid. Cytometry 13:686–692PubMedCrossRefGoogle Scholar
  17. 17.
    Hill CR (1967) Changes in tissue permeability produced by ultrasound. Br J Radiol 40:317–318Google Scholar
  18. 18.
    Honda H, Kondo T, Zhao QL, Feril LB Jr, Kitagawa H (2004) Role of intracellular calcium ions and reactive oxygen species in apoptosis induced by ultrasound. Ultrasound Med Biol 30:683–692PubMedCrossRefGoogle Scholar
  19. 19.
    Jedrzejczak M, Koceva-Chyla A, Gwozdzinski K, Jozwiak Z (1999) Changes in plasma membrane fluidity of immortal rodent cells induced by anticancer drugs doxorubicin, aclarubicin and mitoxantrone. Cell Biol Int 23:497–506PubMedCrossRefGoogle Scholar
  20. 20.
    Kagiya G, Ogawa R, Tabuchi Y, Feril LB Jr, Nozaki T, Fukuda S, Yamamoto K, Kondo T (2006) Expression of heme oxygenase-1 due to intracellular reactive oxygen species induced by ultrasound. Ultrason Sonochem 13:388–396PubMedCrossRefGoogle Scholar
  21. 21.
    Kremkau FW (1979) Cancer therapy with ultrasound: a historical review. J Clin Ultrasound 7:287–300PubMedCrossRefGoogle Scholar
  22. 22.
    Kuypers FA, van den Berg JJ, Schalkwijk C, Roelofsen B, Op den Kamp JA (1987) Parinaric acid as a sensitive fluorescent probe for the determination of lipid peroxidation. Biochim Biophys Acta 921:266–274PubMedGoogle Scholar
  23. 23.
    Loverock P, ter Haar G, Ormerod MG, Imrie PR (1990) The effect of ultrasound on the cytotoxicity of adriamycin. Br J Radiol 63:542–546PubMedCrossRefGoogle Scholar
  24. 24.
    Miller DL, Williams AR, Morris JE, Chrisler WB (1998) Sonoporation of erythrocytes by lithotripter shockwaves in vitro. Ultrasonics 36:947–952PubMedCrossRefGoogle Scholar
  25. 25.
    Murphree SA, Tritton TR, Smith PL, Sartorelli AC (1981) Adriamycin-induced changes in the surface membrane of sarcoma 180 ascites cells. Biochim Biophys Acta 649:317–324PubMedCrossRefGoogle Scholar
  26. 26.
    Nozaki T, Ogawa R, Feril LB Jr, Kagiya G, Fuse H, Kondo T (2003) Enhancement of ultrasound-mediated gene transfection by membrane modification. J Gene Med 5:1046–1055PubMedCrossRefGoogle Scholar
  27. 27.
    Ogawa R, Kagiya G, Feril LB Jr, Nakaya N, Nozaki T, Fuse H, Kondo T (2004) Ultrasound mediated intravesical transfection enhanced by treatment with lidocaine or heat. J Urol 172:1469–1473PubMedCrossRefGoogle Scholar
  28. 28.
    Pagnini U, Pacilio C, Florio S, Crispino A, Claudio PP, Giordano A, Pagnini G (2000) Medroxyprogesterone acetate increases anthracyclines uptake in chronic lymphatic leukemia cells: role of nitric oxide and lipid peroxidation. Anticancer Res 20:33–42PubMedGoogle Scholar
  29. 29.
    Rosenthal I, Sostaric JZ, Riesz P (2004) Sonodynamic therapy- a review of the synergistic effects of drugs and ultrasound. Ultrason Sonochem 11:349–363PubMedGoogle Scholar
  30. 30.
    Saad AH, Hahn GM (1989) Ultrasound enhanced drug toxicity on Chinese hamster ovary cells in vitro. Cancer Res 49:5931–5934PubMedGoogle Scholar
  31. 31.
    Saad AH, Hahn GM (1992) Ultrasound-enhanced effects of adriamycin against murine tumors. Ultrasound Med Biol 18:715–723PubMedCrossRefGoogle Scholar
  32. 32.
    Tata DB, Biglow J, Wu J, Tritton TR, Dunn F (1996) Ultrasound-enhanced hydroxyl radical production from two clinically employed anticancer drugs, adriamycin and mitomycin C. Ultrasonics Sonochem 3:39–45CrossRefGoogle Scholar
  33. 33.
    Tewey KM, Rowe TC, Yang L, Halligan BD, Liu LF (1984) Adriamycin-induced DNA damage mediated by mammalian DNA topoisomerase II. Science 226:466–468PubMedCrossRefGoogle Scholar
  34. 34.
    Umemura S, Yumita N, Okano Y, Kaneuchi M, Magario N, Ishizaki M, Shimizu K, Sano Y, Umemura K, Nishigaki R (1997) Sonodynamically induced in vitro cell damage enhanced by adriamycin. Cancer Lett 121:195–201PubMedCrossRefGoogle Scholar
  35. 35.
    Yu T, Bai J, Hu K, Wang Z (2003) The effect of free radical scavenger and antioxidant on the increase in intracellular adriamycin accumulation induced by ultrasound. Ultrason Sonochem 10:33–35PubMedCrossRefGoogle Scholar
  36. 36.
    Yu T, Wang Z, Jiang S (2001) Potentiation of cytotoxicity of adriamycin on human ovarian carcinoma cell line 3AO by low-level ultrasound. Ultrasonics 39:307–309PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Toru Yoshida
    • 1
  • Takashi Kondo
    • 2
  • Ryohei Ogawa
    • 2
  • Loreto B. FerilJr
    • 2
    • 4
  • Qing-Li Zhao
    • 2
  • Akihiko Watanabe
    • 3
  • Kazuhiro Tsukada
    • 1
  1. 1.Second Department of Surgery, Graduate School of Medicine and Pharmaceutical SciencesUniversity of ToyamaToyamaJapan
  2. 2.Department of Radiological Sciences, 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

Personalised recommendations