We investigated the formation and pharmacology of prostaglandin E 3 (PGE 3 ) derived from fish oil eicosapentaenoic acid (EPA) in human lung cancer A549 cells. Exposure of A549 cells to EPA resulted in the rapid formation and export of PGE 3. The extracellular ratio of PGE 3 to PGE 2 increased from 0.08 in control cells to 0.8 in cells exposed to EPA within 48 h. Incubation of EPA with cloned ovine or human recombinant cyclooxygenase 2 (COX-2) resulted in 13-and 18-fold greater formation of PGE 3 , respectively, than that produced by COX-1. Exposure of A549 cells to 1 M PGE 3 inhibited cell proliferation by 37.1% ( P Ͻ 0.05). Exposure of normal human bronchial epithelial (NHBE) cells to PGE 3 , however, had no effect. When A549 cells were exposed to EPA (25 M) or a combination of EPA and celecoxib (a selective COX-2 inhibitor), the inhibitory effect of EPA on the growth of A549 cells was reversed by the presence of celecoxib (at both 5 and 10 M). This effect appears to be associated with a 50% reduction of PGE 3 formation in cells treated with a combination of EPA and celecoxib compared with cells exposed to EPA alone. These data indicate that exposure of lung cancer cells to EPA results in a decrease in the COX-2-mediated formation of PGE 2 , an increase in the level of PGE 3 , and PGE 3 -mediated inhibition of tumor cell proliferation. 2)]. Epidemiologic studies have shown an inverse relationship between blood levels of n-3 fatty acids derived from fish oils and the risk of prostate and lung cancers (3-5). However, molecular mechanisms for the pharmacologic anticancer activity of EPA have not been fully elucidated. A number of studies have suggested that the anticancer activities of both EPA and DHA are associated with their ability to inhibit the synthesis of 2-series prostaglandins, especially prostaglandin E 2 (PGE 2 ) production [as reviewed in ref. (6)]. In contrast to DHA, however, EPA can actually function as a substrate for COX and result in the synthesis of unique 3-series prostaglandin compounds (7). To date, studies reporting the formation of 3-series prostaglandins by EPA have been performed using normal cells or tissues (8,9). Fischer and Weber (10), for example, provided the first evidence of in vivo formation of thromboxane A 3 and prostaglandin I 3 in humans fed fish oil. In addition, studies conducted in humans have shown that PGE 3 levels increased by ‫ف‬ 10-fold in urine after ingestion of cod liver oil (40 ml/day) for 12 weeks (11).In contrast to PGE 2 , the biological activity of PGE 3 has received little attention. The effect of PGE 3 on cell growth has been reported only in normal murine mammary epithelial (12) and 3T3 fibroblast cells (13). Both studies showed that PGE 2 and PGE 3 stimulated the growth of norAbbreviations: AA, arachidonic acid; BHT, butylated hydroxytoluene; calcein AM, acetoxymethyl ester of calcein; COX, cyclooxygenase; DAPI, 4 Ј ,6-diamidino-2-phenylindole; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; LC/MS/MS, liquid chromatography/tandem mass sp...

Lipid-soluble cardiac glycosides such as bufalin, oleandrin, and digitoxin have been suggested as potent agents that might be useful as anticancer agents. Past research with oleandrin, a principle cardiac glycoside in Nerium oleander L. (Apocynaceae), has been shown to induce cell death through induction of apoptosis. In PANC-1 cells, a human pancreatic cancer cell line, cell death occurs not through apoptosis but rather through autophagy. Oleandrin at low nanomolar concentrations potently inhibited cell proliferation associated with induction of a profound G 2 /M cell cycle arrest. Inhibition of cell cycle was not accompanied by any significant sub G1 accumulation of cells, suggesting a nonapoptotic mechanism. Oleandrin-treated cells exhibited time-and concentration-dependent staining with acridine orange, a lysosomal stain. Subcellular changes within PANC-1 cells included mitochondrial condensation and translocation to a perinuclear position accompanied by vacuoles. Use of a fluorescent oleandrin analog (BODIPYoleandrin) revealed co-localization of the drug within cell mitochondria. Damaged mitochondria were found within autophagosome structures. Formation of autophagosomes was confirmed through electron microscopy and detection of green fluorescent protein-labeled light chain 3 association with autophagosome membranes. Also observed was a drug-mediated inhibition of pAkt formation and up-regulation of pERK. Transfection of Akt into PANC-1 cells or inhibition of pERK activation by MAPK inhibitor abrogated oleandrin-mediated inhibition of cell growth, suggesting that the reduction of pAkt and increased pERK are important to oleandrin's ability to inhibit tumor cell proliferation. The data provide insight into the mechanisms and role of a potent, lipid-soluble cardiac glycoside (oleandrin) in control of human pancreatic cancer proliferation. The use of cardiac glycosides in the treatment of human malignant disease may provide an interesting as well as novel form of therapy. 1-3 For example, oleandrin (Figure 1), the principal cytotoxic component of Nerium oleander, has been shown by several laboratory groups, including our own, to mediate cell death in human but not murine cell lines. 4,5 The mechanisms by which oleandrin selectively controls malignant but not normal cell proliferation may be related to a preferential decreased activation of transcription factors such as nuclear transcription factor-κB (NF-κB) and activator protein-1, 6 alteration of membrane potential and fluidity, 5,7 activation of MAPK and JNK pathways, 7 increased calcineurin content with subsequent FasL expression, 8 up-regulation of death receptors 4 and 5, 9 and induction of reactive oxygen species (ROS) and oxidative stress 10 in tumor cells. Collectively, these mechanisms have been associated with induction of apoptosis and cell death in a wide variety of human tumor cell lines (eg, Jurkat, U-937, HL-60, HeLa, PC3, and MCF-7). Although we also have reported 11 oleandrin-mediated apoptosis in a human prostate cell line (PC3), our recent ...

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Cardiac glycosides such as oleandrin are known to inhibit the Na,K-ATPase pump, resulting in a consequent increase in calcium influx in heart muscle. Here, we investigated the effect of oleandrin on the growth of human and mouse cancer cells in relation to Na,K-ATPase subunits. Oleandrin treatment resulted in selective inhibition of human cancer cell growth but not rodent cell proliferation, which corresponded to the relative level of Na,K-ATPase α3 subunit protein expression. Human pancreatic cancer cell lines were found to differentially express varying levels of α3 protein, but rodent cancer cells lacked discernable expression of this Na,K-ATPase isoform. A correlation was observed between the ratio of α3 to α1 isoforms and the level of oleandrin uptake during inhibition of cell growth and initiation of cell death; the higher the α3 expression relative to α1 expression, the more sensitive the cell was to treatment with oleandrin. Inhibition of proliferation of Panc-1 cells by oleandrin was significantly reduced when the relative expression of α3 was decreased by knocking down the expression of α3 isoform with α3 siRNA or increasing expression of the α1 isoform through transient transfection of α1 cDNA to the cells. Our data suggest that the relative lack of α3 (relative to α1) in rodent and some human tumor cells may explain their unresponsiveness to cardiac glycosides. In conclusion, the relatively higher expression of α3 with the limited expression of α1 may help predict which human tumors are likely to be responsive to treatment with potent lipid-soluble cardiac glycosides such as oleandrin.

The beneficial effects of omega-3 fatty acids are believed to be due in part to selective alteration of arachidonate metabolism that involves cyclooxygenase (COX) enzymes. Here we investigated the effect of eicosapentaenoic acid (EPA) on the proliferation of human non-small cell lung cancer A549 (COX-2 over-expressing) and H1299 (COX-2 null) as well as their xenograft models. While EPA inhibited 50% of proliferation of A549 cells at 6.05 μM, almost 80 μM of EPA was needed to reach similar levels of inhibition of H1299 cells. The formation of PGE3 in A549 cells was almost 3-fold higher than that of H1299 cells when these cells were treated with EPA (25 μM). Intriguingly, when COX-2 expression was reduced by siRNA or shRNA in A549 cells, the antiproliferative activity of EPA was reduced substantially compared to that of control siRNA or shRNA transfected A549 cells. In line with this, dietary menhaden oil significantly inhibited the growth of A549 tumors by reducing tumor weight by 58.8 ± 7.4 %. In contrast, similar diet did not suppress the development of H1299 xenograft. Interestingly, the ratio of PGE3 to PGE2 in A549 was about 0.16 versus only 0.06 in H1299 xenograft tissues. Furthermore, PGE2 up-regulated expression of pAkt, whereas PGE3 downregulated expression of pAkt in A549 cells. Taken together, the results of our study suggest that the ability of EPA to generate PGE3 through COX-2 enzyme might be critical for EPA-mediated tumor growth inhibition which is at least partly due to down-regulation of Akt phosphorylation by PGE3.