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.
Highlights
From the Eicosanoid Biochemistry Section, Laboratory of Molecular Carcinogenesis, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709
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)
The most compelling evidence linking the overexpression of Cox-2 to altered apoptosis and cell differentiation was provided by studies with rat intestinal epithelial (RIE) cells [7]
Summary
Cyclooxygenase; NaBT, sodium butyrate; HETE, hydroxyeicosatetraenoic acid; LO, lipoxygenase; HODE, hydroxyoctadecadienoic acid; RT-PCR, reverse transcriptionpolymerase chain reaction; RIE, rat intestinal epithelial; HpODE, hydroperoxyoctadecadienoic acid; FBS, fetal bovine serum; PBS, phosphate-buffered saline; HPLC, high pressure liquid chromatography; PG, prostaglandin; PPAR, peroxisome proliferator-activated receptor; kb, kilobase(s); bp, base pair(s); NDGA, nordihydroguaiaretic acid. 15-LO metabolites of linoleic acid, 13(S)-HpODE inhibits starvation-induced apoptosis.. Tang et al [14] showed that lipoxygenase metabolites, 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE, inhibit apoptosis in rat W256 cells. They suggest a role for 12-LO as an inhibitor of apoptosis [15]. We have examined arachidonic acid and linoleic acid metabolism in the human colorectal cell line, Caco-2, which undergoes cell differentiation and apoptosis in the presence of NaBT [16]. We show the first evidence that a 15-LO is clearly induced in colorectal carcinoma cells. The 15-LO may act to modulate NaBT-induced apoptosis and cell differentiation in these cells
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