The critical role of 15-lipoxygenase-1 in colorectal epithelial cell terminal differentiation and tumorigenesis

Abstract

Terminal differentiation is an important event for maintaining normal homeostasis in the colorectal epithelium, and the loss of apoptosis is an important mechanism underlying colorectal tumorigenesis. The very limited current data on the role of lipoxygenase (LOX) metabolism in tumorigenesis suggests that the oxidative metabolism of linoleic and arachidonic acid possibly shifts from producing antitumorigenic 15-LOX-1 and 15-LOX-2 products to producing protumorigenic 5-LOX and 12-LOX products. We examined whether this shift occurs in vitro in the human colon cancer cell line Caco-2 in association with the loss of terminal differentiation and apoptosis, or in vivo during the formation of colorectal adenomas in patients with familial adenomatous polyposis (FAP). Restoring terminal differentiation and apoptosis of Caco-2 cells increased the mRNA levels of 5-LOX, 15-LOX-2, and 15-LOX-1, but the only significant increases in protein expression and enzymatic activity were of 15-LOX-1. In FAP patients, 15-LOX-1 expression and activity were significantly down-regulated in adenomas (compared with paired nonneoplastic epithelial mucosa), whereas 5-LOX and 15-LOX-2 protein expressions and enzymatic activities were not. We conducted a validation study with immunohistochemical testing in a second group of FAP patients; 15-LOX-1 expression was down-regulated in colorectal adenomas (compared with nonneoplastic epithelial mucosa) in 87% (13 of 15) of this group. We confirmed the mechanistic relevance of these findings by demonstrating that ectopically restoring 15-LOX-1 expression reestablished apoptosis in Caco-2 cells. Therefore, 15-LOX-1 down-regulation rather than a shift in the balance of LOXs is likely the dominant alteration in LOX metabolism which contributes to colorectal tumorigenesis by repressing apoptosis.

Figure 1

Effects of terminal cell differentiation on lipoxygenase (LOX) expressions and activities. A-C, Caco-2 cells were treated with sodium butyrate (NaBT) and harvested at the indicated times. The expressions of 5-LOX, 15-LOX-1, and 15-LOX-2 were measured using real-time reverse transcription polymerase chain reaction (see Methods). The relative expression levels were calculated as the values relative to that of the calibrator sample (NaBT treated at time 0). Values are the means of triplicate experiments ± standard deviation of the mean (SD). D, Western blots of 5-LOX, 15-LOX-1, and 15-LOX-2, protein expressions in Caco-2 cells were treated as described in Fig. 1A-C. E-G, LOX products during terminal differentiation of Caco-2 cells. Caco-2 cells were treated as described in Fig. 1A-C. Cells and culture medium were extracted at the indicated times, and products were measured using enzyme immunoassays. Values shown are the means ± SDs of triplicate measurements.

Figure 2

Expression and enzymatic activity of lipoxygenases (LOXs) in non-neoplastic mucosa and adenomas of patients with familial adenomatous polyposis (FAP). A, LOX protein expressions in tissues from paired colorectal adenomas and non-neoplastic epithelial mucosa of FAP patients. The adenoma and non-neoplastic mucosa tissues were processed for Western blotting and probed with antibodies for 15-LOX-1, 5-LOX, and 15-LOX-2. Lanes: S, standard positive control of cells transfected with a vector expressing the measured protein; N, non-neoplastic mucosa; P, adenomas. Twenty-five adenomas with paired non-neoplastic mucosa were evaluated; and results of three representative cases are shown. B, LOX products by LC/MS/MS in 25 paired samples of colorectal adenomas (polyp) and their colorectal non-neoplastic epithelial mucosa (normal) from FAP patients. Values are means and standard deviations of the means. C, 15-LOX-1 expression in paired colorectal non-neoplastic epithelial mucosa and adenomas of a second group of FAP patients studied for validation of the primary results shown in panels A and B. Paired samples of 15 patients were processed for 15-LOX-1 immunohistochemical staining, as described in the Methods. Photomicrographs of three representative cases are shown. 15-LOX-1 staining was lower in adenomas of 13 of 15 evaluated patients.

Figure 3

Effects of 15-LOX-1 expression on apoptosis in Caco-2 colon cancer cells. Caco-2 cells were transfected with 15-LOX-1 expression vector, empty vector (control vector), or transfection medium alone (mock transfection), and the effects of 15-LOX-1 expression on cell proliferation and apoptosis were evaluated. A, 15-LOX-1 expression levels in Caco-2 cells with and without 15-LOX-1 transfection. Cells were harvested at the indicated time points after transfection, and 15-LOX-1 expression was assessed by Western blotting. Lanes: S, standard positive control of stably transfected HCT-116 cells with a vector expressing 15-LOX-1; N, negative control; Mock, Caco-2 cells with mock transfection (transfection medium alone); Control Vector, Caco-2 cells transfected with the control (empty) vector; and 15-LOX-1 Vector, Caco-2 transfected with 15-LOX-1 vector. B, Enzymatic activity of the ectopically expressed 15-LOX-1 protein. Caco-2 cells were transfected with medium alone (mock), control vector, or 15-LOX-1 vector and harvested 48 hours following transfection; 13-S-HODE levels were measured by EIA assay. Values are the means of triplicate experiments ± standard deviation of the mean (SD); C, Effects of 15-LOX-1 ectopic expression on cell proliferation. Caco-2 cells were transfected with medium alone (mock), control vector, or 15-LOX-1 vector and then cultured for 96 hours. Live cell counts were performed with trypan blue. Values are the means of triplicate experiments ± SD. D–F, Effects of 15-LOX-1 expression on apoptosis induction in Caco-2 cells. Caco-2 cells were transfected, as described in Fig. 2C and harvested at 96 hours. D, The floating-cell ratio was calculated as a measure of apoptosis. Values are the means of triplicate experiments and SD. E, Apoptosis was evaluated by a DNA laddering assay. Lanes: Ladder, standard DNA ladder; Mock, mock transfected cells; Control Vector, control vector transfected cells; 15-LOX-1 Vector, 15-LOX-1 vector transfected cells. F, Apoptosis was evaluated by assessing PARP protein cleavage with an anti-PARP antibody.