Isolation Of HCC Cancer Stem-like Cells And Chemoresistance Mediated By Akt Pathway

Background and ObjectiveHepatocellular carcinoma (HCC) is the fifth most common cancerworldwide and the third most common cause of cancer mortality. Currently,surgical resection, conventional chemotherapy or radiotherapy is the majormeasure for most patients. Unfortunately, the fact that HCC is resistant tochemotherapy or radiotherapy leaves this disease with no effectivetherapeutic options. Recent years, a small fraction of cancer cells with stemcell properties, named cancer stem cells (CSCs), has been proposed. Thistheory provides a new train of thought for the research and cure of tumor.There is increasing evidence showing that tumours are hierarchicallyorganized and formed, sustained by a very small subpopulation of cancercells called cancer stem cells (CSCs). These cells possess CSCs propertieswith the ability to self-renew and generate the diverse cells that comprisethe tumour and high tumorigenicity in vivo. Based on this theory of CSCs,these cells are responsible for the initiation, recurrence and metastasis ofneoplastic tissue, also for the failure of chemo-and radiotherapy. Therefore,to eradicate a tumor, targeting the CSCs should be performed. This study isto use serum-free medium suspended sphere-forming method to isolate HCCCancer Stem-like Cells from human HCC cell lines and preliminarily discussAkt pathway mediating the sensitivity of liver Cancer Stem-like Cells tochemotherapeutic drug doxorubicin (DOX). Methods1. Human hepatocellular carcinoma (HCC) cell lines PLC、HepG2、Hep3B were cultured in serum-free condition medium to form tumorspheres and PLC cell line was selected to proceed the subsequentexperiments.2. Fluorescence-activated cell sorting (FACS)， colony formationassay，the experiment of inducing multidifferentiation and SCID micetumorigenicity experiments in vivo were used to identify the traits of CSCsin PLC spheres（liver Cancer Stem-like cells）.3. Detect the sensitivity of spheres to chemotherapeutic drug DOXwith MTT and FACS.4. Analyse the variation of apoptosis rate with FACS after joiningDOX and inhibitor specific to PI3K/Akt signaling pathway LY294002toco-incubate spheres.5. Compare the expression of P-AKT1S473at protein levels in PLCspheres with the one in PLC monolayer cells using Western blot and detectthe change of P-AKT1S473、AKT1protein after adding the inhibitorLY294002to PLC spheres.Results1. All of three HCC cell lines could form nonadherent, three-dimensional sphere clusters, called spheres.2. The expression of liver CSCs marker CD90in spheres wasobviously up-regulated as compared to monolayer cells (P<0.01).3. PLC spheres under differentiation serum medium differentiatedlongitudinal stretched non-liver cells4. The coloning number of spheres（123.00±28.48）was2.18times ofthe monolayer cells [（56.33±7.37）(P<0.05)]. 5. The tumorigenicity of spheres was evidently greater than monolayercells when the same number of cells was subcutaneous injected into SCIDmice after7weeks.6. Compared with monolayer cells [proliferation rate:（45.68±5.95）%; apoptosis rate:（41.22±6.73）%], the proliferation rate of spheres（71.83±12.3）%under48hour-treatment of5ug/ml DOX greatly elevated (P<0.05)while the apoptosis rate（11.73±3.77）%sharply reduced (P<0.01). But theapoptosis rate of spheres（35.44±6.65）%sharply increased after addingDOX5ug/ml and LY294002to co-incubate spheres (P<0.01).7. The expression of P-AKT1S473protein in spheres significantlyexceeded monolayer cells (P<0.01) and the P-AKT1S473expression atprotein levels obviously decreased (P<0.05) after adding the inhibitorspecific to PI3K/Akt signaling pathway LY294002to spheres.ConclusionSerum-free medium suspended sphere-forming method couldeffectively enrich liver Cancer Stem-like Cells from HCC cell lines. Theliver Cancer Stem-like Cells demonstrate drug resistance tochemotherapeutic drug DOX and the mechanism is related to Akt signalingpathway molecule AKT1phosphorylated at Ser473. Further research to theinhibitor targeting the molecular is effective to break hepatocellularcarcinoma in clinical.