esculetin and Breast-Neoplasms

Esculetin (6,7-dihydroxycoumarin), a derivative of coumarin compound, is found in traditional medicinal herbs. It has been shown that esculetin triggers diverse cellular signal transduction pathways leading to regulation of physiology in different models. However, whether esculetin affects Ca(2+) homeostasis in breast cancer cells has not been explored. This study examined the underlying mechanism of cytotoxicity induced by esculetin and established the relationship between Ca(2+) signaling and cytotoxicity in human breast cancer cells. The results showed that esculetin induced concentration-dependent rises in the intracellular Ca(2+) concentration ([Ca(2+)]i) in ZR-75-1 (but not in MCF-7 and MDA-MB-231) human breast cancer cells. In ZR-75-1 cells, this Ca(2+) signal response was reduced by removing extracellular Ca(2+) and was inhibited by the store-operated Ca(2+) channel blocker 2-aminoethoxydiphenyl borate (2-APB). In Ca(2+)-free medium, pre-treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) abolished esculetin-induced [Ca(2+)]i rises. Conversely, incubation with esculetin abolished TG-induced [Ca(2+)]i rises. Esculetin induced cytotoxicity that involved apoptosis, as supported by the reduction of mitochondrial membrane potential and the release of cytochrome c and the proteolytic activation of caspase-9/caspase-3, which were partially reversed by pre-chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). Moreover, esculetin increased the percentage of cells in G2/M phase and regulated the expressions of p53, p21, CDK1, and cyclin B1. Together, in ZR-75-1 cells, esculetin induced [Ca(2+)]i rises by releasing Ca(2+) from the ER and causing Ca(2+) influx through 2-APB-sensitive store-operated Ca(2+) entry. Furthermore, esculetin activated Ca(2+)-associated mitochondrial apoptotic pathways that involved G2/M cell cycle arrest. Graphical abstract The summary of esculetin-evoked [Ca(2+)]i rises and -activated Ca(2+)-associated mitochondrial apoptotic pathways that involved cell cycle arrest. The natural coumarin derivative esculetin caused Ca(2+) influx via 2-APB-sensitive store-operated Ca(2+) entry and induced Ca(2+) release from the endoplasmic reticulum. Moreover, esculetin activated the mitochondrial pathway of apoptosis in a Ca(2+)-associated manner that involved G2/M arrest.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Calcium; Calcium Signaling; Cell Cycle Proteins; Cell Line, Tumor; Drug Screening Assays, Antitumor; Female; G1 Phase Cell Cycle Checkpoints; Humans; Inhibitory Concentration 50; Membrane Potential, Mitochondrial; Mitochondria; Umbelliferones

Diets rich in linoleic acid (LA) stimulate the metastasis of MDA-MB-435 human breast cancer cells from the mammary fat pads of nude mice. This omega-6 fatty acid is metabolized to various cyclo-oxygenase and lipoxygenase products, several of which have been previously associated with tumor cell invasion and metastasis. We now report that MDA-MB-435 cells secreted increased levels of prostaglandin E2 (PGE2), and 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE when cultured in the presence of 2.7 microM (0.75 micrograms/ml) LA; 5-HETE secretion was unchanged. The 12-lipoxygenase inhibitor esculetin (20 microM) completely blocked the LA-stimulated 12-HETE secretion. Linoleic acid also increased MDA-MB-435 cell invasion in an in vitro assay; this stimulation was abolished by 20 microM esculetin, but was unaffected by piroxicam, a selective cyclooxygenase inhibitor. The effect of LA on invasion was replicated by 0.1 microM 12-HETE, but not by 5-HETE or PGE2; 15-HETE was stimulatory only at a concentration of 1.0 microM. Zymographic and Northern blot analyses showed that these events are accompanied by the induction of 92 kDa isoform type IV collagenase (metalloproteinase-9) enzymic activity and mRNA expression by exogenous LA and 12-HETE, and their suppression by the 12-lipoxygenase inhibitor. These results suggest that the effects of dietary LA on breast cancer cell metastasis in the nude mouse model are due, at least in part, to enhanced 12-HETE biosynthesis, with an associated increase in proteolytic enzyme activity and tumor cell invasiveness.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Breast Neoplasms; Collagenases; Eicosanoids; Gene Expression; Humans; Hydroxyeicosatetraenoic Acids; Linoleic Acid; Linoleic Acids; Lipoxygenase Inhibitors; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Metastasis; Piroxicam; RNA, Messenger; Tumor Cells, Cultured; Umbelliferones

The effects of inhibitors of arachidonic acid oxidative metabolism, gamma-radiation and/or their combinations on proliferation and cell cycle were studied in human breast carcinoma HS578T and monoblastoid U937 cell lines. While piroxicam an inhibitor of cyclooxygenase pathway, had no significant effects on cell proliferation, inhibitors of lipoxygenase pathway, nordihydroguaiaretic acid and esculetin, suppressed [3H]-thymidine incorporation and cell growth. The latter agents also differed in their modulation of cell cycle parameters depending on the cell line and the time of treatment. When the cells were preirradiated with gamma radiation (5 Gy) and treated with the drugs (at concentrations 50 mumol/l and higher) the effects on cell proliferation were mostly additive. On the other hand, the results suggest that antiproliferative effects could be significantly strengthened when lower doses (25 mumol/l) of lipoxygenase inhibitors were combined with a low dose (1 Gy) of gamma-radiation. Experiments monitoring the reversibility of the effects after single or combined treatment with the agents showed that irradiation suppressed the ability of U937 cells to restore cell proliferation, and that these effects may be strenghtened by esculetin. In conclusion, our results (1) suggest that the lipoxygenase pathway plays a significant role in proliferation of cancer HS578T and U937 cells in vitro, and (2) implicate the possibility of more effective antiproliferative effects after combined treatment of cells with gamma-radiation and lipoxygenase inhibitors.

Topics: Breast Neoplasms; Cell Cycle; Cell Division; Cell Line; Cell Survival; Cyclooxygenase Inhibitors; DNA, Neoplasm; Dose-Response Relationship, Radiation; Female; Gamma Rays; Humans; Kinetics; Lipoxygenase Inhibitors; Masoprocol; Piroxicam; Thymidine; Tumor Cells, Cultured; Umbelliferones

We investigated the effects of piroxicam, esculetin, prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) on a human breast cancer cell line (MDA-MB-231). Both piroxicam and esculetin suppressed cell growth and thymidine incorporation, though esculetin was more active in inhibiting cell growth in the presence of linoleic acid (LA). Esculetin reduced the secretion of LTB independent of LA. Piroxicam reduced the secretion of PGE in the absence of LA but only at higher concentrations in the presence of LA. When the relationship between cell growth and PGE and LTB concentration was evaluated by multivariate regression analysis, cell growth was associated with the PGE and LTB concentration when the cells were treated with esculetin alone or with esculetin and LA. Cell growth was associated only with the PGE concentration when they were treated with piroxicam alone or with piroxicam and LA. Therefore, it appears that the growth of MDA-MB-231 cells in vitro is affected by both lipoxygenase and cyclooxygenase products, though lipoxygenase inhibition is more active than cyclooxygenase inhibition on suppression of cell growth in the presence of LA.

Topics: Breast Neoplasms; Cell Division; Cyclooxygenase Inhibitors; Dinoprostone; Humans; Leukotriene B4; Multivariate Analysis; Piroxicam; Tumor Cells, Cultured; Umbelliferones

Dietary lipids may influence breast cancer progression and prognosis. The MDA-MB-231 human breast cancer cell line was used to examine the direct effects of the various classes of free fatty acids (FAs) on growth in serum-free medium and the involvement of eicosanoid biosynthesis. Linoleic acid, an omega 6 FA, stimulated MDA-MB-231 cell growth with an optimal effect at a concentration of 0.75 microgram/ml, whereas oleic acid, an omega 9 FA, produced growth stimulation at 0.25 microgram/ml but was inhibitory at higher concentrations. Docosahexaenoic acid exhibited a dose-related inhibition of cell growth at concentrations ranging from 0.5 to 2.5 micrograms/ml; eicosapentaenoic acid, also an omega 3 FA, was less effective. Similar inhibitory effects occurred with saturated FAs. Indomethacin, which at high concentrations is an inhibitor of both the cyclooxygenase- and lipoxygenase-catalyzed pathways of eicosanoid synthesis, suppressed cell growth stimulation by an otherwise optimal dose of linoleic acid when present at 40 micrograms/ml. Experiments with piroxicam, nordihydroguaiaretic acid, and esculetin, other inhibitors of eicosanoid biosynthesis with varying selectivity for enzymes of the prostaglandin and leukotriene pathways, indicated that MDA-MB-231 cell growth was dependent on leukotriene rather than prostaglandin production.

Topics: Breast Neoplasms; Cell Division; Eicosanoids; Fatty Acids; Fatty Acids, Unsaturated; Humans; In Vitro Techniques; Indomethacin; Masoprocol; Piroxicam; Tumor Cells, Cultured; Umbelliferones