Doxorubicin and 5-fluorouracil resistant hepatic cancer cells demonstrate stem-like properties

Abstract

The efficacy of hepatocellular carcinoma (HCC) treatment is very low because of the high percentage of recurrence and resistance to anticancer agents. Hepatic cancer stem cells (HCSCs) are considered the origin of such recurrence and resistance. Our aim was to evaluate the stemness of doxorubicin and 5-fluorouracil resistant hepatic cancer cells and establish the new method to isolate the HCSCs from primary cultured HCC tumors. HCC biopsies were used to establish primary cultures. Then, primary cells were selected for HCSCs by culture in medium supplemented with doxorubicin (0, 0.1, 0.25, 0.5 or 1 lg/mL), 5-fluorouracil (0, 0.1, 0.25, 0.5 or 1 lg/mL) or their combination. Selection was confirmed by detection of HCSC markers such as CD133, CD13, CD90, and the side population was identified by rhodamine 123 efflux. The cell population with the strongest expression of these markers was used to evaluate the cell cycle, gene expression profile, tumor sphere formation, marker protein expression, and in vivo tumorigenesis. Selective culture of primary cells in medium supplemented with 0.5 lg/mL doxorubicin and 1 lg/mL 5-fluorouracil selected cancer cells with the highest stemness properties. Selected cells strongly expressed CD13, CD133, CD90, and CD326, efflux rhodamine 123 and formed tumor spheres in suspension. Moreover, selected cells were induced to differentiate into cells with high expression of CD19 and AFP (alpha-fetoprotein), and importantly, could form tumors in NOD/SCID mice upon injection of 1 9 10 5 cells/mouse. Selective culture with doxorubicin and 5-fluorouracil will enrich HCSCs, is an easy method to obtain HCSCs that can be used to develop better therapeutic strategies for patients with HCC, and particularly HCSC-targeting therapy.

Identification of rhodamine 123-positive stem cell subpopulations in canine hepatocellular carcinoma cells

“…In the present study, primary HCCs were obtained from clinical patients; primary HCC were compared with cells cultured with DOX and 5-fluorouracil, which were regarded as HCSCs. HCSCs exhibited more stem cell markers, sphere formation, and tumor differentiation and a lower level of rhodamine expression than primary cells (27). The present study demonstrated that the expression of rhodamine was a poor stem cell marker; however, it did not examine chemoresistance or sphere-forming ability in the Rho Hi and Rho Lo subpopulations (24).…”

“…To the best of our knowledge, there is only one report regarding HCSCs identified by rhodamine expression in humans (27). In the present study, primary HCCs were obtained from clinical patients; primary HCC were compared with cells cultured with DOX and 5-fluorouracil, which were regarded as HCSCs.…”

Mechanisms of doxorubicin resistance in hepatocellular carcinoma

“…The existence of CSCs in human HCC has been experimentally validated through isolation and xenotransplantation assays in immunodeficient mice, and CSCs have proven to have a pivotal role in the development and progression of HCC [114]. CSCs have been shown to be particularly resistant to chemotherapy [115] and cells surviving in the region of trans-arterial embolization-treated tumors display the CSC marker, CD13 [116], suggesting they are a major source of treatment failure and recurrence. Other markers of CSC expression in human HCC, specifically EpCAM and CD133, were found to be increased in TACE-doxorubicin-treated HCC and associated with tumor recurrence after transplant [117].…”

Therapy‐induced enrichment of putative lung cancer stem‐like cells

“…reported that methotrexate treatments as short as 5 days were enough to obtain a cell population of osteosarcoma enriched in CSLC properties. Other recent studies include pancreatic, bladder, liver and laryngeal cancers and lead to the conclusion that drug‐selected cells acquired CSLC characteristics.…”