The first characterization of free radicals formed from cellular COX-catalyzed peroxidation

Abstract

Through free radical-mediated peroxidation, cyclooxygenase (COX) can metabolize dihomo-γ-linolenic acid (DGLA) and arachidonic acid(AA) to form well-known bioactive metabolites, namely, the 1-series of prostaglandins (PGs1) and 2-series of prostaglandins(PGs2), respectively. Unlike PGs2, which are generally viewed as pro-inflammatory and pro-carcinogenic PGs, PGs1 may possess anti-inflammatory and anti-cancer activity. Previous studies using ovine COX along with spin trapping and the LC/ESR/MS technique have shown that certain exclusive free radicals are generated from different free radical reactions in DGLA and AA peroxidation. However, it has been unclear whether the differences were associated with the contrasting bioactivity of DGLA vs. AA. The aim of this study was to refine the LC/MS and spin-trapping technique to make it possible for the association between free radicals and cancer cell growth to be directly tested. Using a colon cancer cell line, HCA-7 colony 29, and LC/MS along with a solid phase extraction, we were able to characterize the reduced forms of radical adducts (hydroxylamines) as the free radicals generated from cellular COX-catalyzed peroxidation. For the first time, free radicals formed in the COX-catalyzed peroxidation of AA vs. DGLA and their association with cancer cell growth was assessed (cell proliferation via MTS and cell cycle distribution via PI staining) in the same experimental setting. The exclusive free radicals formed from the COX-catalyzed peroxidation of AA and DGLA were shown to be correlated with the cell growth response. Our results indicate that free radicals generated from the distinct radical reactions in COX-catalyzed peroxidation may represent the novel metabolites of AA and DGLA that correspond to their contrasting bioactivity.

Free radical derivatives formed from cyclooxygenase-catalyzed dihomo-γ-linolenic acid peroxidation can attenuate colon cancer cell growth and enhance 5-fluorouracil׳s cytotoxicity

“…Meanwhile, a notable accumulation of PGE2 (from COX-catalyzed AA peroxidation) became dominant over the PGE1 and reached ~113 nM at 48 h. The effective accumulation of PGE2 from DGLA-treated cells indicated cellular D5D-mediated conversion of DGLA to AA. This conversion was confirmed by the measurement of much more AA (69.74±8.04) than DGLA (30.93±6.47 nM) in the cells treated with DGLA for 48 h. We also observed the formation of an exclusive AA radical (a carbon–carbon double bond-centered free radical), seen as the hydroxylamine of a POBN adduct with m/z 449, from HCA-7 cells after a 12 h incubation with DGLA [24]. The accumulation of AA as well as the exclusive AA radical at certain time points was also correlated with improved cell proliferation vs. the time points when no or limited AA and PGs-2 were accumulated (data not shown).…”

Anti-cancer activities of ω-6 polyunsaturated fatty acids

“…These studies showed that in addition to a C-15 oxygenation shared by both DGLA and AA, there is a unique C-8 oxygenation pathway present during COX-catalyzed DGLA peroxidation, which can give rise to exclusive DGLA free radicals. [40–42] These studies also demonstrated that the exclusive DGLA free radical derivatives could induce significant inhibition of cell growth and significant cell cycle arrest and apoptosis in the human colon cancer cell line HCA-7 colony 29. [43] Interestingly, in a comparison experiment, PGE1 and PGE2 did not affect cell proliferation at the same concentration, suggesting that the free radicals, rather than PGs, may be more responsible for DGLA’s anti-cancer activity under physiological conditions.…”

“…Recent studies found that DGLA and AA could go through different free radical pathways during lipid peroxidation and produce distinct free radical metabolites. [40–42] It was recently proposed and demonstrated that the adverse effects from ω-6s may be mainly attributed to AA and its metabolite prostaglandin E2 (PGE2), while DGLA may exert an anti-cancer effect via the production of prostaglandin E1 (PGE1) and the exclusive free radical metabolites from its COX-catalyzed lipid peroxidation. [17–23,38,39,43–45] …”

Knockdown delta-5-desaturase promotes the formation of a novel free radical byproduct from COX-catalyzed ω-6 peroxidation to induce apoptosis and sensitize pancreatic cancer cells to chemotherapy drugs

“…Recently, the Qian lab developed a HPLC/ESR/MS combination approach, along with a spin trapping technique, allowing a thorough characterization of commonly and exclusively generated free radicals from COX-catalyzed AA and dihomo-γ-linolenic acid (DGLA, an intermediate precursor of AA) peroxidation [33,34]. A unique structural moiety in DGLA led to the formation of a novel free radical byproduct, 8-hydroxyoctanoic acid (8-HOA) [35]. More recently, the Qian lab also demonstrated that 8-HOA is the bioactive metabolite responsible for DGLA’s anti-cancer effect [35,36].…”

“…A unique structural moiety in DGLA led to the formation of a novel free radical byproduct, 8-hydroxyoctanoic acid (8-HOA) [35]. More recently, the Qian lab also demonstrated that 8-HOA is the bioactive metabolite responsible for DGLA’s anti-cancer effect [35,36]. Direct treatment with certain level of 8-HOA as well as accumulation of 8-HOA from DGLA-treated cells were also found to be essential to inhibit growth of colon cancer cells (e.g., HCA-7 colony 29, overexpresses COX-2) via arresting the cell cycle in G1 and promoting cell apoptosis in a p53-dependent manner [37].…”