Suspension culture combined with chemotherapeutic agents for sorting of breast cancer stem cells.

BACKGROUND: Cancer stem cell (CSC) hypothesis has not been well demonstrated by the lack of the most convincing evidence concerning a single cell capable of giving rise to a tumor. The scarcity in quantity and improper approaches for isolation and purification of CSCs have become the major obstacles for great development in CSCs. Here we adopted suspension culture combined with anticancer regimens as a strategy for screening breast cancer stem cells (BrCSCs). BrCSCs could survive and be highly enriched in non-adherent suspension culture while chemotherapeutic agents could destroy most rapidly dividing cancer cells and spare relatively quiescent BrCSCs. METHODS: TM40D murine breast cancer cells were cultured in serum-free medium. The expression of CD44+CD24- was measured by flow cytometry. Cells of passage 10 were treated in combination with anticancer agents pacilitaxel and epirubicin at different peak plasma concentrations for 24 hours, and then maintained under suspension culture. The rate of apoptosis was examined by flow cytometry with Annexin-V fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining method. Selected cells in different amounts were injected subcutaneously into BALB/C mice to observe tumor formation. RESULTS: Cells of passage 10 in suspension culture had the highest percentage of CD44+CD24- (about 77 percent). A single tumor cell in 0.35 PPC could generate tumors in 3 of 20 BALB/C mice. CONCLUSION: Suspension culture combined with anticancer regimens provides an effective means of isolating, culturing and purifying BrCSCs.

Suspension culture combined with anticancer regimens for screening breast cancer stem cells.

Breast cancer stem cells (BrCSCs)have been first identified in solid tumors. To be more exact, they are defined as tumorigenic cancer cells which exhibit the capacity to form tumors distinct from the majority non-tumorigenic cancer cells. However it is only a minor subset that cancer stem cells are highly enriched in. It is still unknown how to find the definitive research subject - "a single breast cancer stem cell" - that can cause a new tumor by itself. Since recent studies suggest that BrCSCs have a higher proportion in suspension culture and have a greater resistance to chemotherapy regimens, we propose a novel strategy to isolate and identify BrCSCs:suspension culture combined with anticancer regimens. This double sorting method by means of the unique properties of BrCSCs may be helpful to screen genuine cancer stem cells(CSCs).

Caspase-8 in apoptosis of hepatoma cell induced by 5-fluorouracil.

OBJECTIVE: To explore the relationship between the changes in the activity of caspase-8 and apoptosis of HepG2 cells induced by 5-fluorouracil (5-Fu). METHODS: Human hepatoma HepG2 cells were treated with 5-Fu at the concentrations of 1X10(-1), 1X10(-2), 1X10(-3), 1X10(-4), 1X10(-5) mol/L and for 1, 2, 4, 8, 16, 24 hours, respectively. The caspase-8 activity was detected using caspase-8 fluorescent assay kit. The apoptotic rate of HepG2 cells induced by 5-Fu with or without the caspase-8 inhibitor IETD-FMK was measured by flow cytometry. RESULTS: After the HepG2 cells were treated with 10(-2) mol/L 5-Fu, the caspase-8 activity increased gradually and reached the peak level (313.9+/-6.9) at 16 hours, then fell down. Compared with the control group, the activity was still significantly higher (274.2+/-3.9 vs 68.3+/-3.6, P<0.01). With the increasing concentration of 5-Fu, the caspase-8 activity was also increased; the activity in high concentration 5-Fu was significantly higher than that in low concentration 5-Fu (370.5+/-4.7 vs 313.7+/-6.9; 225.7+/-5.4 vs 183.3+/-4.8; 183.3+/-4.8 vs 124.0+/-6.2, P<0.01). The caspase-8 activity was the highest at 1X10(-1) mol/L 5-Fu (370.5+/-4.7). The caspase-8 activity in low concentration 5-Fu was higher than in the blank control group and inhibitor group (124.0+/-6.2 vs 68.5+/-3.4; 124.0+/-6.2 vs 41.0+/-2.1, P<0.01). IETD-FMK could block the activation of caspase-8 and reduce the apoptosis of HepG2 cells induced by 5-Fu. The apoptotic rate of HepG2 cells in the 5-Fu group was significantly different from that in the inhibitor group (P<0.01). CONCLUSIONS: 5-Fu can induce apoptosis of HepG2 cells via caspase-8 signal transduction pathway, which can be blocked by IETD-FMK. 5-Fu promotes the increase of caspase-8 activity in a time- or concentration-dependent manner.