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
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
复制标题
DOI：
10.1007/s00280-007-0503-y
复制
发表时间：
2008-04-01
影响因子：
3
通讯作者：
Tsukada, Kazuhiro
中科院分区：
文献类型：
--
作者：
Yoshida, Toru;Kondo, Takashi;Tsukada, Kazuhiro
目的超声（US）在增强抗癌药物治疗癌症的效果方面的潜在临床应用已在先前的报告中得到强调。由于US，癌细胞对药物的摄取增加已被认为是一种机制。然而，增强的确切机制尚未阐明。本研究探讨低强度脉冲超声联合阿霉素（DOX）对U937细胞的杀伤和诱导凋亡作用及其机制。实验在4组中进行：（1）未处理的，（2）DOX处理的（DOX），（3）US处理的（US），和（4）组合的（DOX + US）。在DOX + US中，将细胞暴露于5 μ M DOX 30 min，并通过1 MHz脉冲US（PRF 100 Hz，DF 10%）以0.2-0.5 W/cm（2）的强度超声处理60 s。洗涤细胞并孵育6小时。台盼蓝拒染法检测细胞存活率，流式细胞仪检测细胞凋亡和DOX掺入。用FITC-葡聚糖评价声孔在分子掺入中的作用，用电子顺磁共振-自旋捕获法测定羟自由基的形成，用顺式-帕里纳酸和二萘嵌苯荧光偏振法评价DOX引起的膜改变，包括脂质过氧化和膜流动性，结果在0.3W/cm（2）及以上的功率密度下，可观察到细胞杀伤的协同增强作用和诱导凋亡的累加增强作用。在0.2 W/cm（2）时，未观察到细胞杀伤和诱导凋亡的增强。在0.3 W/cm（2）及以上时检测到羟基自由基形成。在DOX + US中产生的自由基多于单独的US。在0.5 W/cm（2）下，DOX + US（与DOX相比）中DOX的掺入增加了13%。利用FITC标记的葡聚糖进行的实验表明，声孔效应参与了药物摄取的增加。我们假设DOX处理使细胞对US的机械作用减弱。5 μ M的DOX处理30 min对细胞膜的脂质过氧化和流动性无明显影响，但随着DOX浓度的增加和处理时间的延长，细胞膜的脂质过氧化和流动性发生了明显的变化。结论DOX增强细胞膜脂质过氧化和流动性的机制可能是：（1）超声促进DOX的掺入，与声孔效应有关;空化是增强DOX效应的必要条件。虽然DOX对膜修饰的确切参与仍有待阐明，但它们可能参与了潜在效应。
Purpose 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 mu M 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 mu M 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.Conclusion 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.