| Doxorubicin(DOX),an anthracycline antineoplastic agent which is the cornerstone of many chemotherapeutic protocols,has been widely utilized in cancer treatment due to its efficacy in fighting a broad spectrum of malignancies.Despite its effectiveness,the DOX-induced side effects are often regarded as the major problem,limiting its clinical use.In recent decades,microbubble(MB)-mediated ultrasound(US)therapy has been proposed as an innovative method for non-invasive delivery of bioactive agents into tumor cells,potentially serving to lower the required dosage amounts and minimize the adverse events of drugs.Thus,this novel therapy with the combination of antitumor drugs and MB-US stands out from the rest.Based on the previous work of our research team,we aimed to investigate the combined efficacy of low-level focused US,DOX and a clinically approved MB,SonoVue on in vitro cultUred human leukemia K562 cells.By detecting the physiobiochemic and hemorheological indexes of cell death,DNA damage,etc.,we explored the mechanism of DOX + MB-US-induced K562 cell injury,paving the way for the future clinical use of Sonochemotherapy(SCT).Our research findings obtained so far are as follows:1.From the results of MTT assay,ultrasound inhibited the cell viability of K562 cells in both ultrasound intensity-and cell density-dependent manner,and the former was much obvious than the latter;DOX inhibited K562 cells proliferation in a dose-,incubation time-and cell density-dependent manner.The cell density of 2×105 cells/ml might be a sensitive threshold for the DOX-induced cell killing on K562 cells;The combined therapy with DOX and US inhibited K562 cells proliferation depending on DOX dose,ultrasound intensity and incubation time.The cytotoxicity of DOX might be efficiently enhanced via US application with optimized parameters;Noticeably,with the administration of MB into the US field,a remarkable synergistic increment on cell proliferation inhibition was detected in the combined groups at various US intensity.Wherein,the synergistic effects at 3 W were more significant;Moreover,Guava Viacount assay was adopted to examine cell viability.The results demonstrated that our optimized US application system could activate MB and then affect cell viability.2.As determined by TA dosimetry,the generation of ·OH radicals resulting from cavitation was greatly enhanced with MB administration;By measuring changes in FD500-uptake and FDA-efflux through flow cytometry,a consistent conclusion has been reached that the MB-US treatment significantly enhanced the cellular membrane permeabilization,compared with US alone treatment group.Moreover,based on the data from the detection of intracellular DOX levels,we found that DOX-uptake was obviously increased in MB-US treatment group when compared with the others.Such data indicate that our optimized US application system could activate MBs,enhance the cavitation effect and aggravate cell membrane damages,facilitating the cellular uptake of DOX molecules and resulting in the subsequent biological responses.3?Apoptosis was analyzed using Annexin V-PE/7-amino-actinomycin D staining.A positive enhancement on necrotic cell death was detected at 24 h after the combined treatment with DOX and MB-US.4?DNA fragment assay suggested that DOX + MB-US treatment significantly potentiated DNA fragmentation,while more damaged cells with DNA morphological changes were observed in the same group as demonstrated by DAPI staining.These results reveal that the synergistic effects on DNA damage occurred when treated with the combination of DOX and MB-US in K562 cells.5?According to the data from flow cytometry,a significant increase on intracellular reactive oxygen species production and an obvious decline in mitochondrial transmembrane potential after treated with DOX and MB-US,implying that ROS may be involved in the SCT-induced cell injury,while mitochondria may act as one of the sensitive targets in it. |
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