Intestinal bacterium-derived cyp27a1 prevents colon cancer cell apoptosis

Abstract

The pathogenesis of metastasis of colon cancer (Cca) is to be further investigated. The dysfunction of apoptotic mechanism plays a role in the cancer cell over growth. This study tests a hypothesis by which intestinal bacterium-derived cyp27a1 prevents apoptosis in colon cancer cells. In this study, the levels of cyp27a1 in human stool samples were assessed by enzyme-linked immunosorbent assay. The apoptosis of Cca cells was observed by flow cytometry. The expression of cyp27a1 was assessed by real time RT-PCR and Western blotting. We observed higher levels of cyp27a1 in the stool samples of Cca patients than that from healthy subjects. Cca colon epithelial biopsy contained high levels of cyp27a1 protein, but not the cyp27a1 mRNA. Cyp27a1 prevented Cca cell apoptosis induced by vitamin D3. In conclusion, intestinal bacterium-derived cyp27a1 facilitates Cca survival by inhibiting Cca cell apoptosis.

Keywords: Colon cancer; Cyp27a1; apoptosis; vitamin D.

Figure 1

Cca patient stool bacteria express high levels of cyp27a1. A: The bars indicate the cyp27a1 protein levels (by ELISA) in stool samples of 32 Cca patients and 20 healthy subjects. B and C: The bars indicate the mRNA levels of cyp27a1 (B: By RT-qPCR) and the immune blots indicate the protein levels of cyp27a1 (C: By Western blotting) in bacteria isolated from the stool samples. Data are presented as mean ± SD. *, P<0.01, compared with healthy subjects. Samples from individual patients were analyzed separately. Antibody of cyp27a1 was purchased from Santa Cruz Biotec (Santa Cruz, CA).

Figure 2

Colon epithelial cells absorb cyp27a1 from the intestinal tract. A: The immune blots indicate the cyp27a1 protein levels in colon epithelial samples (analyzed by Western blotting). The samples were collected from 20 non-cancer subjects and 20 Cca patients. B: The bars indicate the integrated density of the immune blots of panel A (summarized from all the readouts of the individual samples). C: The bars indicate the cyp27a1 mRNA levels of the samples (analyzed by RT-qPCR). Data of bars are presented as mean ± SD. *, P<0.01, compared with the non-cancer group.

Figure 3

Cyp27a1 interferes with the effect of VD3 on suppression of cancer cell growth. The bars indicate the number of T84 cancer cells at time points from 0 hr to 144 hr. Control: T84 cells were cultured in the presence of PMA (40 ng/ml). VD3: In the presence of VD3 (10 nM) and PMA (Phorbol 12-myristate 13-acetate; Sigma Aldrich). VD3/cyp27a1: In the presence of VD3, PMA and cyp27a1 (10 nM). Data of bars are presented as mean ± SD. *, P<0.01, compared with the control group. VD3 was purchased from Sigma Aldrich (St. Louis., MO). Cyp27a1 was provided by Sangon Biotech (Shanghai, China).

Figure 4

Cyp27a1 inhibits Cca cell apoptosis. (A-E) The gated dot plots indicate the frequency of apoptotic Cca cells. The Cca cells (T84 cells) were cultured for 3 days. The culture condition is denoted on the X xis of (F) (the summarized data of apoptotic cells in A-E). The cells were stained with Annexin V and propidium iodide, and analyzed by flow cytometry. VD3: 10 nM. Cyp27a1: 10 nM. Cyp27b1-d: cyp27b1-deficient Cca cells (generated by RNAi as shown by G).