Taken together, these results suggested that SOX2 regulated population growth and properties of CSCs in colorectal cancer following irradiation, and SOX2 may be a potential target for studies involving resistance to radiation

Taken together, these results suggested that SOX2 regulated population growth and properties of CSCs in colorectal cancer following irradiation, and SOX2 may be a potential target for studies involving resistance to radiation. Open in a separate window Figure 3 Knockdown of SOX2 in radioresistant colorectal cancer cells attenuated the induction of colorectal CSCs after irradiation. radioresistant cells. Further analysis revealed the retention of CSC properties with an upregulation of SOX2 as shown by enhanced resistance to radiation and metastatic potential in vitro. Interestingly, both the knockdown and overexpression of SOX2 led to increase in CD44+ population and induction of CSC properties in colorectal cancer following irradiation. Furthermore, selective genetic and pharmacological inhibition of the PI3K/AKT pathway, but not the MAPK pathway, attenuated SOX2-dependent CD44 expression and metastatic potential upon irradiation in vitro. Our findings suggested that STAT2 SOX2 regulated by radiation-induced activation of PI3K/AKT pathway contributes to the induction of colorectal CSCs, thereby highlighting its potential as a therapeutic target. = 3) with * < 0.05 for the pairwise comparisons between radioresistant cells and radiosensitive cells. (B) Colony formation assay was performed with indicated cells treated with 4 Gy (left panel). Graph showing quantification of relative colony numbers at different doses of IR (right panel). (C) Cell populations for the CD44+, CD133+, or ALDH+, which are known markers of cancer stem-like cells (CSC) in these indicated cells after radiation exposure were measured by flow cytometric analysis. The percentage of each CSC marker-expressing cell is shown as a bar graph. Data are shown as mean SD (= 3) with * < 0.05 for the pairwise comparisons between radioresistant cells and radiosensitive cells. (D) Cells were stained Hoechst 33342 analog 2 with an anti-CD44 antibody (green) and anti-CD133 antibody (red). Nuclei were counterstained with DAPI ((blue). CSCs: cancer stem-like cells. 3.2. Radiation-Enriched CD44+ Cells Exhibited the Properties of CSCs Including an Increase in SOX2 Expression To delineate the role of radiation-induced CD44 expression in radioresistant colorectal cancer cells, we isolated both CD44 positive (CD44+) and negative (CD44?) cells in HCT116 and DLD1 cells following irradiation using anti-CD44-FITC antibodies by FACS, and the expression of CD44 in both CD44+ and CD44? cells is shown in Figure 2A. Since the CD44 marker correlated with the features of CSCs in colorectal cancers [19,20], we evaluated the properties of colorectal CSCs including metastatic potential and self-renewal. We observed an increase in colony formation, migration and invasion in the sorted CD44+ cells after irradiation and not in CD44? cells in both cell lines (Figure 2BCD). Interestingly, immunoblotting of stemness-related proteins revealed significant elevation in SOX2 levels among stemness-related proteins [21,22] on sorted CD44+ cells (Figure 2A). Given the evidence that SOX2 was aberrantly expressed and involved in the maintenance of CSCs in colorectal cancer [14,15], these results indicated the possibility of a functional relationship between SOX2 expression and CD44-mediated CSC property in radioresistant cells upon radiation exposure. Open in a separate window Figure 2 CD44+ cells induced by radiation exhibited the properties of cancer stem-like cells (CSCs) with an increase in SOX2 levels. (A) CD44+ CD44? cells on day 2 after irradiation with 10 Gy in radioresistant colorectal cancer cells (HCT116 and DLD1) were sorted (left panel). Immunoblotting for Hoechst 33342 analog 2 the expression of CSC-related proteins in CD44+ (positive) and CD44? (negative) in radioresistant cells (right panel). (B) Colony formation assay was performed with CD44+ (or CD44?) cells, and the bar graphs show the quantification of relative colony numbers in indicated cells. Data are shown as mean SD (= 3) * < 0.05 compared to control. (C,D) The migration and invasion analysis (left panel) and quantification of cells involved in migration and invasion Hoechst 33342 analog 2 (right panel) in CD44+ and CD44? cells sorted from HCT116 and DLD1 cells, respectively. All experiments were performed in triplicates. Data are shown as mean SD. * < 0.05 compared to CD44? cell. CD44?: negative, CD44+: positive, CSCs: cancer stem-like cells. 3.3. Modulation of SOX2 Expression in Colorectal Cancer Cells Is Associated with Induction of Colorectal CSCs Following Irradiation We further determined.