Mechanisms of Arachidonic Acid In Vitro Tumoricidal Impact

Abstract

To promote the potential of arachidonic acid (ARA) for cancer prevention and management, experiments were implemented to disclose the mechanisms of its tumoricidal action. Hepatocellular, lung, and breast carcinoma and normal hepatocytes cell lines were exposed to 0 or 50 μM ARA for 30 min and then assessed for proliferative capacity, surface membrane-associated sphingomyelin (SM) content, neutral sphingomyelinase (nSMase) activity, beta 2 microglobulin (β2 m) expression, and ceramide (Cer) levels. Reactive oxygen species (ROS) content and caspase 3/7 activity were evaluated. Exposure to ARA for 30 min led to impairment of the tumor cells' proliferative capacity and revealed that the different cell lines display remarkably similar surface membrane SM content but diverse responses to ARA treatment. Arachidonic acid tumoricidal impact was shown to be associated with nSMase activation, exposure of cell surface membrane β2 m to antibody binding, and hydrolysis of SM to Cer, which accumulated on the cell surface and in the cytosol. The ARA and Cer-mediated inhibition of tumor cell viability appeared to be independent of ROS generation or caspase 3/7 activation. The data were compared and contrasted to findings reported in the literature on ARA tumoricidal mechanisms.

Mechanisms of Arachidonic Acid In Vitro Schistosomicidal Potential

“…The D-PBS containing Triton-soluble and insoluble surface membrane molecules ,, was aspirated, assessed for protein content, distributed into aliquots to be maintained at −20 °C, and thawed only once.…”

“… 33 After washing, control and lysenin-exposed worms were incubated with 1:100-diluted rabbit antibodies to lysenin (MyBioSource, San Diego, CA, USA), washed 3× and incubated with 1:100-diluted fluorescein isothiocyanate (FITC)-labeled antibodies to rabbit immunoglobulins (Sigma), and inspected by alternate light and UV microscopy. 71 …”

“…After washing in medium/1% FCS, all worms were incubated for 1 h at room temperature with 1:50-diluted FITC-labeled antibody to mouse IgG (H+L) (Sigma), examined, and photographed under light and ultraviolet microscopy. 71 …”

“…Duplicate 20 μg protein aliquots of surface membrane and homogenate extracts of untreated and ARA-exposed worms were evaluated for nSMase activity using a Sphingomyelinase Assay Kit of Abcam, ab287874 (Cambridge Biomedical Campus, Cambridge, United Kingdom), following the manufacturer’s instructions. 71 Fluorescence was measured at Ex/Em of 540/590 nm at 15, 30, and 60 min after adding reagents, and counts were shown after subtracting mean background values (Victor X4Multi-Label Plate Reader, PerkinElmer, Waltham, MA, USA).…”

“…Lysenin, a protein derived from the coelomic fluid of the earthworm Eisenia fetida, recognizes the SM heterogeneous organization in bio-membranes via specific binding to clusters of 5–6 SM molecules . After washing, control and lysenin-exposed worms were incubated with 1:100-diluted rabbit antibodies to lysenin (MyBioSource, San Diego, CA, USA), washed 3× and incubated with 1:100-diluted fluorescein isothiocyanate (FITC)-labeled antibodies to rabbit immunoglobulins (Sigma), and inspected by alternate light and UV microscopy …”