MC3 Mucoepidermoid carcinoma cell line enriched cancer stem-like cells following chemotherapy

Abstract

Mucoepidermoid carcinoma (MEC) is common in human salivary glands. Surgery is the preferred treatment method for MEC and chemotherapy is often administered following surgery as an adjuvant cancer treatment; however, chemotherapy does not completely prevent tumor recurrence. Emerging evidence has indicated the existence of cancer stem-like (CSL)-cells in tumors. CSL-cells are important in the development, invasion and drug resistance of carcinomas. The present study aimed to investigate whether chemotherapy enriched the CSL-cells in the MEC cell line of MC3 using 5-fluorouracil (5-Fu). The MC3 cells were treated with 5-Fu, which enhanced the spherogenesis and vitality of the cells and upregulated the pluripotency gene, octamer-binding transcription factor 4. Side population analysis demonstrated that the proportion of CSL-cells also increased. These findings showed that compared with other types of cancer cells, chemotherapy was unable to effectively kill the CSL-cells resulting in an enriched CSL-cell subpopulation with a higher resistance to chemotherapy, which may have been key the recurrence of MEC.

Keywords: 5-fluorouracil; cancer stem-like cells; cluster of differentiation 44; mucoepidermoid carcinoma; octamer-binding transcription factor 4.

Figure 1

Cloning ratio of 5-Fu (drug)-treated and parent MC3 cells in soft agarose. The cloning ratio of 5-Fu-treated cells was significantly higher than the parent MC3 cells (P<0.05). 5-Fu, 5-fluorouracil.

Figure 2

Growth curves of 5-Fu (drug)-treated cells and parent MC3 cells, the proliferative ability of 5-Fu-treated cells was significantly higher than the parent MC3 cells (P<0.05). 5-Fu, 5-fluorouracil; OD, optical density.

Figure 3

Gene expression examined by quantitative polymerase chain reaction. The expression of CD44 and Oct4 was statistically different between the 5-Fu (drug)-treated and parent MC3 cells (P<0.05). 5-Fu, 5-fluorouracil; Oct4, octamer-binding transcription factor 4; CD44, cluster of differentiation 44. ^*P<0.05 parent MC3 cells vs. 5-Fu treated cells

Figure 4

Immunocytochemistry assays of Oct4 and CD44 in (A) 5-Fu-treated and (B) parent MC3 cells. The expression of CD44 and Oct4 in the 5-Fu-treated cells was higher than in the parent MC3 cells. (C) Expression of Oct4 and CD44 in the isotype control. 5-Fu, 5-fluorouracil; Oct4, octamer-binding transcription factor 4; CD44, cluster of differentitation 44.

Figure 5

FACS analysis of Oct4 and CD44 in the (A) blank control, (B) parent MC3 and (C) 5-Fu-treated cells. The percentage of Oct4+ and CD44+ phenotype in the parent MC3 cells was 1.37±0.06 and 14.47±0.15%, respectively. The percentage of Oct4+ and CD44+ phenotype in the 5-Fu-treated cells was 14.60±0.36% and 99.50±0.30%, respectively. FACS, fluorescence-activated cell sorting; 5-Fu, 5-fluorouracil; Oct4, octamer-binding transcription factor 4; CD44, cluster of differentitation 44; FITC-A, fluorescein isothiocyanate labeled antibody.

Figure 6

Spheroids of (A) 5-Fu (drug)-treated and (B) parent MC3 cells in serum-free medium. (C) The cloning (spheroids) ratio of 5-Fu-treated and MC3 cells was 44.02±1.71 and 19.94±0.57%, respectively (P<0.05). 5-Fu, 5-fluorouracil.

Figure 7

FACS analysis of (A) 5-Fu-treated and (B) parent MC3 cells stained with Hoechst 33342. The ratio of side population cells in the 5-Fu-treated and MC3 cells was 8.3±0.2 and 2.03±0.25%, respectively. FACS, fluorescence-activated cell sorting; 5-Fu, 5-fluorouracil.