Objective      To isolate hepatocellular carcinoma (HCC) cancer stem-like cells and investigate the role of PI3K/Akt pathway in the sensitivity of liver cancer stem-like cells to chemotherapeutic drug doxorubicin (DOX).        Methods      Human HCC cell lines PLC

HepG2

and Hep3B were cultured in serum-free condition medium to form tumor spheres

and PLC cell line was finally selected to proceed the subsequent experiments. Fluorescence-activated cell sorting (FACS) and colony formation assay and SCID mice tumorigenicity experiments in vivo were used to identify the traits of CSCs in PLC spheres（liver cancer stem-like cells）. The sensitivity of the cancer stem-like cells to DOX was detected with MTT assay and FACS. The apoptotic rate of the cancer stem-like cells was analyzed with FACS after the treatment of DOX and LY294002

an inhibitor specific to PI3K/Akt signaling pathway. Western blotting was used to detect the expression of p-Akt1Ser473 and Akt1 protein in PLC spheres and in PLC monolayer cells in present or absent of the inhibitor LY294002.        Results      The expression of liver CSCs marker CD90 in the obtained spheres was obviously up-regulated as compared to monolayer cells (P0.01). The cloning number of spheres （123.00±28.48）was 2.18 times higher than that of the monolayer cells（56.33±7.37

P0.05). The tumorigenicity of spheres was evidently greater than the monolayer cells when same number of cells was subcutaneous injected into SCID mice after 7 weeks. The proliferation rate was greatly elevated after 48 hour-treatment of 5 μg/mL DOX in the spheres than in the monolayer cells [(71.83±12.30）% vs （45.68±5.95）%

P0.05]

while the apoptosis rate was sharply reduced [（11.73±3.77）% vs （41.22±6.73）%

P0.01]. But the apoptotic rate of spheres was sharply increased after the treatment of 5 μg/mL DOX and LY294002. （35.44±6.65）%

P0.01). The expression of p-Akt1Ser473 protein significantly exceeded in spheres than monolayer cells (P0.01) and the p-Akt1Ser473 expression at protein levels was obviously decreased after addition of LY294002 (P0.05). There was no change in the expression of Akt1 protein (P0.05).        Conclusion      The liver cancer stem-like cells demonstrate drug resistance to chemotherapeutic drug DOX

and the mechanism is related to Akt signaling pathway molecule Akt1 phosphorylated at Ser473.