Phenotypic characteristics of CD133+ EpCAM+ cancer stem-like cells derived from the human hepatoma HepG2 cell line

Abstract

Background/aim Cancer stem-like cells (CSCs) have been found to be a serious hurdle in the effective treatment of cancer. The rationale of this study was to isolate and characterize CD133+EpCAM+-enriched cells from the human hepatoma HepG2 cell line to prove their stemness phenotype. Materials and methods The CD133+EpCAM+ cells were sorted from the HepG2 cell line using magnetic cell sorting and specified by flow cytometry analysis of surface markers [CD13, CD24, CD34, CD44, CD90, CD133, and CD326 (EpCAM)] and transmission electron microscopy to confirm their identity as CSCs. Quantitative real-time PCR analysis was applied for determining the expression level of stemness marker genes: Oct4, Nanog, ALDH1A1, Notch receptors (NOTCH1, NOTCH2, and NOTCH3), and cytokeratins (CK8/18/19). The proliferative ability of the isolated cells was identified through MTT assay, and their sensitivity to chemotherapeutic drugs was measured by cell counting kit-8 assay. Results The isolated CD133+EpCAM+ cells from the HepG2 cell line characterized by flow cytometry were positive for CD13 (81.8%), CD24 (24.4%), CD34 (3.36%), CD44 (92.0%), CD90 (39.7%), CD133 (82.3%), and CD326 (2.79%). Moreover, our data clarified from transmission electron microscopy examination that the isolated CD133+EpCAM+ cells exhibited irregular cell morphology and integral cell membrane structure. The sorted CD133+EpCAM+ cells possessed considerable increase in the mRNA level of Oct4, Nanog, ALDH1A1, NOTCH1, NOTCH2, NOTCH3, and CK19 genes, whereas they showed significant decrease in the mRNA level of CK8 and CK18 genes versus CD133-EpCAM- cells. Moreover, starting from day 4 to day 10, the CD133+EpCAM+ cells showed a significant increase in their proliferation rate and displayed high resistance to chemotherapy (doxorubicin) contrary to CD133-EpCAM- cells. Conclusion On the basis of the aforementioned results, CD133+EpCAM+-enriched cells strictly represented CSC phenotype in the HepG2 cell line. These cells might be valuable for studying the mechanism of CSCs in hepatoma and screening novel targets for cancer therapy.