Altered characteristics of cancer stem/initiating cells in a breast cancer cell line treated with persistent 5-FU chemotherapy

Abstract

Drug resistance of cancer stem/initiating cells has been considered to be one of the main reasons for tumor relapse. However, knowledge concerning the changes in stem/ initiating cells during chemotherapy is limited. In the present study, the breast cancer cell line MDA-MB-468 was cultured with 5-fluorouracil and serially passaged. Six cell generations were collected. Semi-quantitative RT-PCR and flow cytometric techniques were used to evaluate the protein and mRNA expression of stem/initiating factors (CD44(+)/CD24(-), Oct 3/4, SOX2 and β-catenin), drug-resistance genes (BCRP and MRP1) and an anti-apoptosis gene (survivin). The clone formation rate was also examined in every generation of cells. The results showed that, under conditions of persistent chemotherapy, the factors representing the quantity of stem/initiating cells (β-catenin, Oct 3/4 and SOX2) followed a fluctuating trend of decrease-increase-further increase-decrease-increase-decrease, and factors representing the proportion of stem/initiating cells (proportion of CD44(+)/CD24(-) and the clone formation rate) demonstrated a fluctuating trend of increase-further increase-further increase-decrease. The drug-resistance genes (BCRP and MRP1) and the anti-apoptosis gene (survivin) demonstrated a wave of increase-further increase-further increase-decrease-increase (MRP1 decrease)-decrease. β-catenin, Oct 3/4 and SOX2 showed a positive correlation (r=1, p<0.01). Our study confirmed that the drug resistance of cancer cells is mainly due to tumor stem/initiating cells, and that under conditions of persistent chemotherapy, the quantity or function of breast cancer stem/initiating cells increases and decreases alternately.

Figure 1.

Trend of mRNA expression of the relative markers in each cell generation in the control group. The x-axis indicates cell generation, whereas the y-axis indicates the relative value of mRNA expression.

Figure 2.

Electrophorogram of mRNA expression of the relative markers in each cell generation in the experimental group. M, molecular weight marker; the left marker is the 100-bp ladder, the right marker is DL2000 (apart from Oct 3/4, whose marker was the 100-bp ladder). C, control group. Lanes 1, 2, 3, 4, 5 and 6 represent the first, second, third, fourth, fifth and sixth cell generation, respectively.

Figure 3.

Trend of mRNA expression of the relative markers in each cell generation in the experimental group. The x-axis indicates the cell generation, whereas the y-axis indicates the relative value of mRNA expression.

Figure 4.

Proportion of CD44⁺/CD24⁻ cells in the experimental group. Images 1, 2, 3, 4 and 5 are the proportion of CD44⁺/CD24⁻ cells in the non-treated group, the first, second, third and fourth generation of cells, respectively. The CD44⁺/CD24⁻ cells were located in the upper left quadrant. Image 6 is a line graph showing the fluctuation in the proportion of CD44⁺/ CD24⁻ cells in different cell generations. The x-axis indicates the cell generation, whereas the y-axis denotes the percentage.

Figure 5.

Colony formation in the experimental group. Images 1, 2, 3, 4 and 5 show the colony formation in the non-treated group, and first, second, third, fourth generation cells, respectively. Image 6 is a line graph showing fluctuations in the colony forming rate (colonies/10⁴ seeding cells) in the different cell generations. The x-axis indicates the cell generation, whereas the y-xis denotes the quantity of colonies.