Deficiency in the 15 kDa selenoprotein inhibits human colon cancer cell growth

Abstract

Selenium is an essential micronutrient for humans and animals, and is thought to provide protection against some forms of cancer. These protective effects appear to be mediated, at least in part, through selenium-containing proteins (selenoproteins). Recent studies in a mouse colon cancer cell line have shown that the 15 kDa selenoprotein (Sep15) may also play a role in promoting colon cancer. The current study investigated whether the effects of reversing the cancer phenotype observed when Sep15 was removed in mouse colon cancer cells, were recapitulated in HCT116 and HT29 human colorectal carcinoma cells. Targeted down-regulation of Sep15 using RNAi technology in these human colon cancer cell lines resulted in similarly decreased growth under anchorage-dependent and anchorage-independent conditions. However, the magnitude of reduction in cell growth was much less than in the mouse colon cancer cell line investigated previously. Furthermore, changes in cell cycle distribution were observed, indicating a delayed release of Sep15 deficient cells from the G(0)/G(1) phase after synchronization. The potential mechanism by which human colon cancer cells lacking Sep15 revert their cancer phenotype will need to be explored further.

Keywords: HCT116 cells; HT29 cells; cancer prevention; selenium; shRNA.

Figure 1

mRNA expression of selenoproteins in Sep15 deficient human colorectal cancer cells. mRNA levels of (a) Sep15, (b) GPx1, (c) GPx2 and (d) TR1 were measured using quantitative real-time RT-PCR, and GAPDH was used as an internal control. Data are displayed as means ± SE (ANOVA, n = 3) and were graphed relative to expression in HCT116 control cells (cycle threshold values were 17.6, 20.1, 19.6, 29.7 and 23.5 for GAPDH, SEP15, GPX1, GPX2 and TR1, respectively).

Figure 2

Protein expression of Sep15, GPx1/2 and TR1 upon targeted removal of Sep15 in HCT116 and HT29 human colorectal cancer cells, as determined by labeling cells with ⁷⁵Se.

Figure 3

Effects of Sep15 deficiency on cell growth over a four day period with (a) HCT116 cells and (b) HT29 cells. Values are means ± SE (t-tests, n = 3–6).

Figure 4

Effects of Sep15 deficiency on anchorage-independent colony formation in soft agar as quantitated by staining cell colonies with p-iodonitrotetrazolium overnight. (a) Representative soft agar dishes with stained colonies of HT29 cells after 20 days; numbers of colonies of (b) HCT116 cells and (c) HT29 cells. Values are mean ± SE (t-test, n = 4).

Figure 5

Cell cycle analysis of HCT116 and HT29 cells. Percent of cells in each phase of the cell cycle as determined by FACS analysis at six hours after release from synchronization with serum is shown. Values are means ± SE (t-tests, n = 6).

Figure 6

mRNA expression of guanylate binding protein-1 (GBP-1) and interferon-γ (IFN-γ). mRNA levels of (a) GBP-1 and (b) IFN-γin HCT116 and HT29 colon cancer cells were determined by quantitative real-time RT-PCR, with GAPDH used as internal control. Columns depict means ± SE (t-test, n = 6), and were graphed relative to expression in HCT116 control cells (cycle threshold values were 17.6, 22.3 and 31.5 for GAPDH, GBP-1 and IFN-γ, respectively).