Expression of 15-Lipoxygenase by Human Colorectal Carcinoma Caco-2 Cells during Apoptosis and Cell Differentiation

Abstract

We studied arachidonic acid metabolism and the expression of cyclooxygenase (Cox) and 15-lipoxygenase (15-LO) in the human colorectal carcinoma cell line, Caco-2, which undergo apoptosis and cell differentiation in the presence of sodium butyrate (NaBT). Caco-2 cells expressed very low levels of Cox-1 but highly expressed Cox-2. NaBT treatment shifted the arachidonic acid metabolites by cell lysates from prostaglandins to 15-hydroxyeicosatetraenoic acid, indicating the presence of a 15-LO. Linoleic acid, an excellent substrate for 15-LO, was metabolized poorly by the Caco-2 cells, but NaBT treatment shifted metabolism to 15-LO metabolite, 13(S)-hydroxyoctadecadienoic acid. Caco-2 cells expressed a 15-LO but only after treatment with NaBT, as determined by Northern blotting. Immunoblotting with anti-human 15-LO antibody detected a 72-kDa band in NaBT-treated Caco-2 cells. Expression of 15-LO mRNA was dependent on the duration of NaBT treatment, with the highest expression observed between 10 and 24 h. Results from expression and metabolism studies with arachidonic and linoleic acid cells indicated Cox-2 was responsible for the lipid metabolism in control cells, whereas 15-LO was the major enzyme responsible after NaBT induction of apoptosis and cell differentiation. The 15-LO in Caco-2 cells was characterized as human reticulocyte 15-LO by reverse transcription-polymerase chain reaction and restriction enzyme analysis. The expression of 15-LO and 15-hydroxyeicosatetraenoic acid or 13(S)-hydroxyoctadecadienoic acid formation correlates with cell differentiation or apoptosis in Caco-2 cells induced by NaBT. The addition of nordihydroguaiaretic acid, a lipoxygenase inhibitor, significantly increased NaBT-induced apoptosis, whereas the addition of indomethacin did not alter NaBT-induced apoptosis in the Caco-2 cells. However, indomethacin treatment decreased the expression of Cox-2 in NaBT-treated cells and significantly increased the expression of 15-LO during NaBT treatment. These studies suggest a role for 15-LO, in addition to Cox-2, in modulating NaBT-induced apoptosis and cell differentiation in human colorectal carcinoma cells.The first genetic alteration in the multistep process that leads to the development of colon carcinogenesis seems to be the loss of APC gene function. Investigations of APC function and its mutations have provided important clues in understanding colon cancer (1). One of the important changes that results from the loss of the APC gene function is the overexpression of prostaglandin H synthase-2 (cyclooxygenase-2, Cox-2), 1 an inducible enzyme that converts arachidonic acid to prostaglandins. The link between APC, Cox-2, and polyp formation was firmly established in studies using APC (Apc ⌬716 ) and Cox-2 knockout mice (2). Mice carrying the APC mutation overexpress Cox-2 and develop intestinal polyps. When bred to mice with the disrupted Cox-2 gene, the offspring, homozygously deficient for Cox-2, had significantly less polyps than the mice with wild-type Cox-2. These results confirmed C...

15-Lipoxygenase-1 Metabolites Down-regulate Peroxisome Proliferator-activated Receptor γ via the MAPK Signaling Pathway

“…In human colorectal carcinoma Caco-2 cells, sodium butyrate induces the expression of reticulocyte 15-LO-1, and these cells undergo differentiation and apoptosis (5). This study provided the first evidence that 15-LO-1 is clearly expressed in human colorectal carcinoma cells, and it was subsequently shown that the 15-LO-1 is uniquely regulated by histone acetylation (6).…”

IGF-II/IGF-I receptor pathway up-regulates COX-2 mRNA expression and PGE2 synthesis in Caco-2 human colon carcinoma cells

“…We used Caco-2 cells as a model system to study the mechanisms that regulates COX-2 expression in colon cancer cells. Caco-2 cells express COX-2, but not COX-1, mRNA levels, expression being variable in di erent studies probably due to di erent clones of Caco-2 cells used and/or di erent experimental conditions (Kutchera et al, 1996;Tsujii et al, 1997Tsujii et al, , 1998Kamitani et al, 1998).…”

Lipoxygenases and their involvement in programmed cell death

“…11 Therefore, it is not surprising that activation of lipoxygenases has been associated to programmed death of different cells and tissues, induced by different, unrelated stimuli (Table 1 and references therein). Several studies have shown the proapoptotic activity of 5-LOX, 15,23 of leukocyte type 12-LOX, 21,26 and of 15-LOX, 24,28 and have indicated molecular targets for lipoxygenase interaction able to contribute to the induction of apoptosis (Table 2 and references therein). However, also anti-apoptotic effects of LOXs have been reported, mainly based on the observation that LOX inhibitors, most often nordihydroguaiaretic acid (NDGA) and MK886, had pro-apoptotic activity.…”