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Objective To investigate the cytotoxicity of paclitaxel combined with ultrasound irradiation on three-dimentional spheroids and explore the related mechanisms.Methods The human breast cancer MCF-7 cell and human nasopharyngeal carcinoma 5-8F cell were used to establish the three-dimentional (3D) spheroids in serum-free medium.The difference of cell cycle and multidrug resistance associated protein (MRP) expression between 3D spheroids and 2D monolayer cells were determined by flow cytometry,Q-PCR and CCK-8 experiments.In addition, CCK-8 test was used to evaluate the cell viability of 3D spheroids and 2D monolayer cells with or without ultrasound (US) irradiation in the optimized parameter.Difference of intercellular space of each spheroid was determined by H&E staining, while the hypoxia marker HIF-1α was detected by Q-PCR analysis.Moreover,proteins related to drug resistance or hypoxia of 3D spheroids after US exposure were estimated by western blot analysis.Results The 3D MCF-7 and 5-8F spheroid with the characteristic of drug resistant and hypoxia were successfully established.Compared to the group of paclitaxel without US,the cytotoxicity of paclitaxel significantly increased when combined with US at high-dose paclitaxel (<1 ng/ml for MCF-7 spheroids and <2 ng/ml for 5-8F spheroids,P<0.05), indicating that US played an important role in enhancing the cytotoxicity of paclitaxel.H&E staining showed that the intercellular space of MCF-7 spheroids was wider than that of 5-8F, while expression of HIF-1α was significantly lower than that of 5-8F determined by Q-PCR (P<0.001).Compared to control group, P-gp, BCRP, MRP1, VEGF expression levels of 5-8F spheroids were down-regulated after US.Expression level of HIF-1α protein did not change.P-gp expression level of MCF-7 spheroids increased slightly after US, and BCRP, MRP1, HIF-1α, VEGF expression levels did not change significantly.It indicated that protein expression levels were different in various spheroids in response to US exposure.The DNA damage marker γ-H2AX was significantly increased at 72 h after US for both MCF-7 and 5-8F spheroids.Conclusion US can enhance the cytotoxity of paclitaxel for MCF-7 and 5-8F spheroids,which might be not only attributed to the enhancement of cell membrane permeability generated by US, and the difference of intracellular space that facilitating drug penetration into inner area of the spheroids, but also the downregulation of drug-resistant and hypoxic proteins and inducement of DNA damage.