Isolation of sub-clones from parent HT29 cells and expression of RAS, RAF and MAPK pathway proteins in HT29 sub-clones
To investigate the heterogeneity of the cell line, we established sub-clones from HT29 colon cancer cell line. We prepared sub-clones using a traditional cloning cylinder method. The morphology of sub-clones showed a round shape and clustered growth, and almost no difference was observed among clones. However, the growth rate was quite different among sub-clones with a full confluency time ranging from 20 to 32 h (Figure 1A).
The clone cell stock took 7 to 21 days to prepare. The doubling time was different for each clone. We used western blotting to analyze heterogeneous protein expression in RAS, RAF and MAPK pathway in sub-clones (Figure 1B), which are activated for cell growth, division and differentiation . The expression of EGFR was higher in HT29s01, HT29s02, HT29s06, and HT29s08 than in parental cell line, and the KRAS expression was higher in HT29s03 but differed between each clone. The expression of BRAF, MEK, and ERK was lower in HT29s03, HT29s05, HT29s07, and HT29s12 than in other clones.
HT29 cells are EGFR positive, containing wild-type KRAS and mutant BRAF proteins . In practice, it is considered that EGFR-positive as well as KRAS wild-type cells have differences in downstream signaling. The results showed differential expression in the BRAF/MEK/ERK pathway. Six clones (HT29s01, HT29s02, HT29s06, HT29s08, HT29s10, HT29s11 cells) exhibited a relatively high level of protein expression than other clones (HT29s03, HT29s05, HT29s07, HT29s09).
The doubling time of each sub-clone
Distinct subpopulations expressed cell growth pathway proteins, as shown in Figure 1B. We next analyzed the growth rate of eleven clones using the MTT assay. Approximately 4,000 cells/well were seeded in a 96-well plate and allowed to attach for 24, 48, and 72 h (Figure 1A). The doubling time of parental HT29 cells was 23 h, whereas, for each sub-clone, it was different. Growth rates were: 20.3 h for HT29s01 cells; 20.9 h for HT29s02 cells; 29.6 h for HT29s03 cells; 26.25 h for HT29s04 cells; 31.8 h for HT29s05 cells; 20.9 h for HT29s06 cells; 28.6 h for HT29s07 cells; 19.3 h for HT29s08 cells; 24 h for HT29s09 cells; 21.1 h for HT29s10 cells; 21.1 h for HT29s11 cells . Heterogeneous growth of the cells in each clone was observed. We divided the cells into two groups according to the doubling time: Fast-growing group (HT29s01, HT29s02, HT29s08, HT29s10, HT29s11(red lines)), and slow-growing group (HT29s03, HT29s04, HT29s05, HT29s07 (blue lines)).
Oxaliplatin Cytotoxicity in HT29 sub-clones
Cancer stem cells exhibited a decrease in cellular proliferation. Cells were treated with various concentrations of oxaliplatin for 72 h to investigate oxaliplatin cytotoxicity in HT29 sub-clones (Figure 2). Oxaliplatin had a different effect on each HT29 sub-clone. HT29 parental cells had an IC50 value of 0.53 μM. HT29s01 (IC50 0.12 μM), HT29s02 (IC50 0.15 μM), HT29s04 (IC50 0.58 μM), HT29s06 (IC50 0.11 μM), HT29s08 (IC50 0.12 μM), HT29s09 (IC50 0.62 μM), HT29s10 (IC50 0.13 μM) and HT29s11 (IC50 0.16 μM) were more sensitive than HT29s03 (IC50 4.8 μM), HT29s05 (IC50 11.1 μM) and HT29s07 (IC50 3.69 μM). Parental cells and the fast-growing group were sensitive to oxaliplatin. However, HT29s03, HT29s05 and HT29s07 cells were resistant to oxaliplatin after 72 h of exposure. In the previous data (Figure 1A), the slow-growing group had significantly higher chemoresistance than the fast-growing group. Oxaliplatin resistant cells showed the same features as cancer stem cells.
The response of sensitive and resistant cells to oxaliplatin treatment in vivo
The clones were classified as oxaliplatin sensitive or resistant according to their sensitivity to the oxaliplatin treatment in vitro. However, the sensitivity of cancer cells is not always correlated with in vivo experiments. Therefore, we examined the in vivo response of oxaliplatin in nude mice. The clones, which had similar growth rates, were selected from the oxaliplatin-sensitive (HT29s04) and -resistant group (HT29s05). Further, 2×106 cells were subcutaneously inoculated into nude mice flank and oxaliplatin treatment (10 mg/kg, once a week, for 30 days) followed 2 weeks later. The average final tumor volume of HT29s04 was 164 ± 148.6 mm3 in the control group and 46.2 ± 12.8 mm3 in the oxaliplatin treatment group. Furthermore, the average final tumor volume of HT29s05 was 442.7 ± 324.7 mm3 in the control group, and 385.1 ± 123.4 mm3 in the oxaliplatin treated group (Figure 3). The treatment of HT29s05 with oxaliplatin did not show a significant difference (p>0.05) in tumor growth, but a dramatic decrease of tumor growth was observed in HT29s04, which was in the oxaliplatin sensitive group. These data are consistent with the results of the in vitro cytotoxicity assay.
Expression of ABC transporter in HT29 sub-clones
Drug transporters are an important mechanism of chemotherapy resistance. The ABC drug transporters were shown to protect cancer stem cells from chemotherapeutic agents. We examined the protein levels of ABC transporters in the sub-clones using by western blot analysis (Figure 4).
The overexpression of ABC transporters in cancer is considered to be a primary determinant of the multidrug-resistant (MDR) phenotype. We detected the expressions of three primary ABC transporters (ABCB1, ABCC2, ABCG2) by western blot analysis to find whether the resistant cells showed drug resistance to oxaliplatin through these ABC transporters. ABCG2 showed low expression, whereas ABCB1 showed mostly high expression. However, ABCC2 was expressed in high levels in HT29s03, HT29s05, HT29s07 and HT29s09 cells, but not in HT29s04 cells, among the slow-growing group when compared to the fast-growing group.