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AIM: To investigate the multicellular resistance of human hepatocellular carcinoma HepG2 cells in three-dimensional culture to delisheng, 5-fluorouracil and adriamycin, and the possible molecular mechanisms of delisheng.METHODS: Human.hepatocellular carcinoma HepG2 cells were cultured with a liquid overlay technique. After the formation of multicellular spheroids, morphology was analyzed by phase contrast microscopy, scanning electron microscopy and transmission electron microscopy.Sensitivity of HepG2 cells to delisheng, 5-fluorouracil and adriamycin was investigated by MTT assay in multicelluar spheroids and monolayers. Vascular endothelial growth factor (VEGF) and endostatin expression were analyzed in multicellular spheroids treated with delisheng,5-fluorouracil, adriamycin and negative control PBS, with immunohistochemical staining.RESULTS: Multicellular spheroids exhibited structural characteristics somewhat different to those in monolayers. The cells in three-dimensional cell culture ted out to be less sensitive to delisheng, 5-fluorouracil and adriamycin than the cells cultured in monolayer. This showed that delisheng had a satisfactory cells inhibition ratio compared to 5-fluorouracil and adriamycin.Immunohistochemical staining showed that VEGF and endostatin expression was positive during growth as multicellular spheroids, and endostatin expression in spheroids with treatment of delisheng was higher than that with 5-fluorouracil, adriamycin and PBS (139.35±7.83, 159.23±10.34, 162.83±3.47 and 148.48 ± 11.06, P＜0.05).CONCLUSION: Chinese medicine compound delisheng has satisfactory anti-tumor activity in HepG2 cells in three-dimensional culture, and the effects are associated with up-regulation of endostatin.