Slug expression inhibits calcitriol-mediated sensitivity to radiation in colorectal cancer

Abstract

Recently, a reciprocal relationship between calcitriol and epithelial-to-mesenchymal transition has been described. Therefore, we hypothesized that calcitriol (1α,25-dihydroxyvitamin D₃) would enhance radiation sensitivity in colorectal cancer regulated by epithelial mesenchymal transition. Vitamin-D receptor, E-cadherin and vimentin protein as well as E-cadherin, Snail and Slug mRNA levels were assessed in a panel of human colorectal cancer cell lines at baseline and in response calcitriol. We defined cell lines as calcitriol sensitive based on demonstrating an enhanced epithelial phenotype with increased E-cadherin, reduced vimentin and decreased expression of Snail and Slug as well as decreased cellular migration in response to calcitriol. In calcitriol sensitive cells, including DLD-1 and HCT116, 24 h calcitriol pre-treatment enhanced the radiation sensitivity by 2.3- and 2.6-fold, respectively, at 4 Gy (P < 0.05). In contrast, SW620 cells with high baseline mesenchymal features including high Slug and vimentin expression with low E-cadherin expression demonstrated no significant radiation sensitizing response to calcitriol treatment. Similarly, transfection of Slug in the calcitriol sensitive colon cancer cell lines, DLD-1 and HCT 116, completely inhibited the radiation sensitizing effect of calcitriol. Collectively, we demonstrate that calcitriol can enhance the therapeutic effects of radiation in colon cancer cells and Slug expression mitigates this observed effect potentially representing an effective biomarker for calcitriol therapy.

Keywords: EMT; Slug; cancer; colorectal; radiation; vitamin D.

Figure 1

Variable expression of EMT markers and Vitamin D receptor in a panel of colon cancer cell lines. (A) Western Blot analysis of vimentin, E-cadherin, and VDR from total cell lysates of various colon cancer cell lines. GAPDH was used as a loading control. (B) Real-time RT-PCR analysis of E-cadherin, VDR, Slug, and Snail mRNA expression normalized to GAPDH.

Figure 2

Calcitriol treatment augments expression of E-cadherin in colon cancer cells. (A) Western Blot analysis of E-cadherin in DLD1, HCT116, and SW620 colon cancer cells treated with 1 μM calcitriol for the indicated time in hours. GAPDH was used as a loading control. (B) Immunofluorescent E-cadherin staining after 48 h of 1 μM calcitriol (right panels) or vehicle control (left panels) treatment.

Figure 3

Calcitriol treatment alters expression of Snail and Slug. Real-time RT-PCR analysis of Snail (left panels), Slug (middle panels), and Vimentin (right panels) from (A) DLD-1, (B) HCT116, and (C) SW620 colon cancer cells treated with 1 μM calcitriol for the indicated time in hours. *P < 0.05, **P < 0.005.

Figure 4

Calcitriol pre-treatment variably regulates colon cancer cellular migration. DLD-1, HCT116, and SW620 colon cancer cells were pre-treated with 1 μM calcitriol (gray bars) or vehicle alone (black bars) for 24 and 48 h, then plated on Boyden migration chamber inserts. Cellular migration was quantified as mean number of cells migrating through the transwell inserts. Columns, average % of cells invading per high-powered field. *P < 0.02.

Figure 5

Calcitriol sensitizes colon cancer cells to radiation. DLD-1, HCT116, and SW620 colon cancer cells were pre-treated with 1 μM calcitriol (gray bars) or vehicle (black bars) for 24 h prior to radiation treatment of 0–6 Gy. After 10 d, surviving colonies of 20 or more cells were counted to determine the % survival relative to the untreated cells. *P < 0.01, **P < 0.05.

Figure 6

Overexpression of Slug inhibits calcitriol’s radiation sensitizing ability. (A) Real-time RT-PCR confirmed expression of Snail and Slug in stable transfected pools of both Snail and Slug overexpressing DLD-1 (black bars) and HCT116 (gray bars) cells compared to the empty vector (EV) controls. Data were normalized to GAPDH. (B) Western Blot analysis of VDR and E-cadherin from total cell lysates of Snail and Slug expressing HCT116 and DLD-1 cells compared to the empty vector controls. GAPDH was used as a loading control. (C) Cell lines were pre-treated with 1 μM calcitriol (gray bars) or vehicle alone (black bars) for 24 h prior to radiation treatment of 0–6 Gy and compared with empty vector stably transfected cells. After 10 d, surviving colonies of 20 or more cells were counted to determine the % survival relative to the untreated cells. *P < 0.01, **P < 0.05.

Figure 7

Model of calcitriol mediated sensitivity to radiation. In DLD-1 and HCT116 cells, Slug is a negative regulator of E-cadherin. The addition of calcitriol leads to repression of Slug and a subsequent increase in E-cadherin (E Cad) and/or membrane localized E-cadherin resulting in an increased sensitivity to radiation. However, in SW620 cells in which both Slug and vimentin (Vim) are induced with the addition of calcitriol, calcitriol therapy does not increase E-cadherin levels and therefore we see no change in radiation sensitivity.