title: 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.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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 microM DOX for 30 min and sonicated by 1 MHz pulsed US (PRF 100 Hz, DF 10%) at intensities of 0.2-0.5 W/cm(2) 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.
  
  
  RESULTS
  Synergistic enhancement in cell killing and additive enhancement in induction of apoptosis were observed at and above 0.3 W/cm(2). No enhancement was observed at 0.2 W/cm(2) in cell killing and induction of apoptosis. Hydroxyl radicals formation was detected at and above 0.3 W/cm(2). 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/cm(2). 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 microM 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Second Department of Surgery, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 930-0194, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Yoshida
authors.forename: Takashi
authors.initials: T
authors.lastname: Kondo
authors.forename: Ryohei
authors.initials: R
authors.lastname: Ogawa
authors.forename: Loreto B
authors.initials: LB
authors.lastname: Feril
authors.forename: Qing-Li
authors.initials: QL
authors.lastname: Zhao
authors.forename: Akihiko
authors.initials: A
authors.lastname: Watanabe
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Tsukada
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000378:metabolism / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects* / Q000528:radiation effects*; D016923:Cell Death / Q000187:drug effects / Q000528:radiation effects; D002462:Cell Membrane / Q000187:drug effects / Q000648:ultrastructure; D003911:Dextrans; D004317:Doxorubicin / Q000378:metabolism / Q000633:toxicity*; D004578:Electron Spin Resonance Spectroscopy; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D005456:Fluorescent Dyes; D005609:Free Radicals / Q000378:metabolism; D006801:Humans; D017665:Hydroxyl Radical / Q000378:metabolism; D015227:Lipid Peroxidation / Q000187:drug effects; D008560:Membrane Fluidity / Q000187:drug effects; D020298:U937 Cells; D014465:Ultrasonics
chemical_list: D000903:Antibiotics, Antineoplastic; D003911:Dextrans; D005456:Fluorescent Dyes; D005609:Free Radicals; C015219:fluorescein isothiocyanate dextran; D017665:Hydroxyl Radical; D004317:Doxorubicin; D016650:Fluorescein-5-isothiocyanate