Arachidonic acid metabolite

12-HETE activates the ILK/NF-κB pathway, which contributes to the suppression of cell apoptosis and suggests a key underlying mechanism that sustains the viability of ovarian cancer cells. 12-HETE promotes cell survival in ovarian cancer via triggering the integrin-linked kinase/NF-κB pathway. 12-HETE exhibits concentration-dependent protection against ovarian cancer cells' apoptosis. 12-HETE (1 µM) dramatically reduces the serum deprivation (SD)-induced caspase-3 activation. With an IC50 value of 1.13 µM, 12-HETE represses the increased activity of caspase-3 produced by SD in a concentration-dependent manner[1]. In ovarian cancer cells, 12-HETE (1 µM) promotes NF-κB activation and nuclear translocation via ILK[1]. 12-HETE causes cell death in human islets, lowers metabolic activity, and decreases insulin secretion. 12-HETE suppresses prostaglandin E1-induced elevation of intracellular cAMP levels while enhancing thrombin-induced bovine platelet aggregation. 12-HETE prevents the aggregation of washed platelets (WP)[2]. 12-HETE has a number of effects on neurons, including a reduction in glutamate release and calcium influx as well as a suppression of AMPA receptor (AMPA-R) activation[3].

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In our study, we found that 12-HETE, a major metabolic product of arachidonic acid using 12-LOX catalysis, inhibited cell apoptosis in a dose-dependent manner and that the effects of 12-HETE on cell apoptosis were mediated by the integrin-linked kinase (ILK) pathway. Moreover, the downstream target of 12-HETE-activated ILK was nuclear factor kappa-B (NF-κB) in ovarian carcinoma. The inhibitory effects of 12-HETE on cell apoptosis were attenuated by the inhibition of the NF-κB pathway.
Conclusion: These results indicate that 12-HETE participates in the inhibition of cell apoptosis by activating the ILK/NF-κB pathway, implying an important underlying mechanism that promotes the survival of ovarian cancer cells. [1]

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12-HETE inhibits cell apoptosis in a dose-dependent manner [1]
It is well known that an increased resistance to apoptosis is a hallmark of cancer. To determine the crucial role of 12-HETE in the growth of ovarian cancer cells, we examined whether 12-HETE protected against cell apoptosis in ovarian cancer cells. In this study, SD was used as an apoptotic model for experiments. As shown in Figure 1A, the MTT results showed that cell viability was significantly decreased by SD after 48 hours of treatment, while SD-induced decreases in cell viability were obviously mitigated by 1 µM exogeneous 12-HETE. We obtained similar results when we detected the activity of caspase-3. The results show that 1 µM 12-HETE significantly decreases the activation of caspase-3 induced by SD (Figure 1B). Moreover, we also examined cellular half maximal inhibitory concentration (IC50) values of 12-HETE for the caspase-3 activity in OVCAR-3 cells and found that 12-HETE repressed the increased activity of caspase-3 induced by SD in a concentration-dependent manner, with an IC50 value of 1.13 µM (Figure 1C). The results indicate that 12-HETE protects against cell apoptosis in ovarian cancer cells in a concentration-dependent manner.

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The effects of 12-HETE on cell apoptosis are mediated by the ILK pathway [1]
Our previous study demonstrated that ILK participates in the regulation of the progression of ovarian cancer and plays a critical role in the survival of cancer cells.15 We examined whether 12-HETE was responsible for the activation of the ILK pathway during the progression of ovarian cancer. In OVCAR-3 cells, treatment with 1 µM 12-HETE led to the increased expression of ILK (Figure 2A). To elucidate the role of ILK in the inhibition of cell apoptosis by 12-HETE, ILK siRNA (siILK) was used to knock down the expression of ILK (Figure 2B). As shown in Figure S1, cell viability, caspase-3 activity, and ILK expression were not obviously affected by the transfection of siControl compared with the untransfected OVCAR-3 cells (Figure S1A–C), implying that there are no significant differences between the siControl transfection and untransfected cells. Thus, we utilized the siControl transfection cells as the negative control in the following experiments. The results of a cell growth assay showed that 1 µM 12-HETE antagonized the decrease in cell viability induced by SD, but the effects of 12-HETE on cell survival were abolished after repressing the expression of ILK with siILK (Figure 2C). As shown in Figure 2D, the release of LDH (an indication of cell death) induced by SD was attenuated by 1 µM 12-HETE, whereas the ILK siRNA eliminated the roles of 12-HETE in LDH release (Figure 2D). Moreover, the inhibitory effects of 12-HETE on the activation of caspase-3 and the increased expression of cleaved caspase-3 induced by SD were attenuated by ILK siRNA (Figure 2E and F). These results imply that 12-HETE promotes the survival of ovarian cancer cells by activating the ILK pathway.

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12-HETE activation of the ILK pathway inhibits mitochondria-dependent apoptosis [1]
Intrinsic cell apoptosis is an important event induced by SD that leads to cell growth arrest. We examined some important changes in mitochondria-dependent apoptosis, including the protein levels of Bcl-2 and Bax, the release of cytochrome C into the cytoplasm, and the activity of caspase-9. As shown in Figure 3A and B, 1 µM 12-HETE induced the expression of Bcl-2 (an antiapoptotic protein) and depressed the expression of Bax (a pro-apoptotic protein), and these effects were weakened by the ILK siRNA. Moreover, the release of cytochrome C from the mitochondria into the cytoplasm is a critical event that triggers the progression of mitochondria-dependent apoptosis. We found that SD treatment led to increased expression of cytoplasmic cytochrome C and decreased expression of cytochrome C in mitochondria, but 1 µM 12-HETE mitigated the release of cytochrome C from the mitochondria into the cytoplasm. However, the inhibitory effects of 12-HETE on cytochrome C release were eliminated by the ILK siRNA (Figure 3C). In addition, we also examined the activity of caspase-9, which is specifically activated in the intrinsic apoptosis pathway. We found that 1 µM 12-HETE inhibited the activation of caspase-9 induced by SD, while the ILK siRNA impaired the inhibitory effects of 12-HETE on caspase-9 activation (Figure 3D). These results imply that ILK is involved in the inhibition of mitochondria-dependent apoptosis by 12-HETE.
Meanwhile, to validate the role of 12-HETE in cell apop-tosis, we also tried an additional concentration of 12-HETE for some functional experiments. Our results showed that the treatment with 3 µM 12-HETE induced the expression of ILK in OVCAR-3 cells (Figure S2A). Both the decreased cell viability and the increased LDH release induced by SD were mitigated by 3 µM 12-HETE. However, the ILK siRNA antagonized the facilitating roles of 12-HETE in cell survival (Figure S2B and C). Moreover, the treatment with 3 µM 12-HETE inhibited the activation of caspase-3 and increased the expression of Bax induced by SD, but these effects were eliminated by ILK siRNA (Figure S2D and E).

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The activation of NF-κB induced by 12-HETE is eliminated by silencing the expression of ILK [1]
NF-κB is an ubiquitous transcription factor the dysfunction of which is closely related to different types of cancers. The increased activation of NF-κB is a predictor of poor disease progression and confers resistance to cell apoptosis.16 Based on this evidence, we examined the status of NF-κB in ovarian cancer cells under conditions of 12-HETE treatment. The results showed that 1 µM 12-HETE led to increased levels of NF-κB p65 phosphorylation, and the increased phosphorylation of NF-κB p65 caused by 12-HETE was attenuated by the knockdown of ILK (Figure 4A). Moreover, the protein levels of NF-κB p65 in cytoplasmic and nuclear extracts indicated that 1 µM 12-HETE treatment caused a significant increase in the protein levels of nuclear NF-κB p65, which was accompanied by decreased levels of NF-κB p65 in the cytoplasm. However, the nuclear translocation elicited by 12-HETE treatment was abolished by the ILK siRNA (Figure 4B). In addition, the activation of NF-κB is closely modulated by IKB kinases. Hence, we further examined the effects of 12-HETE on the phosphorylation of IKBa protein. As shown in Figure 4C, 1 µM 12-HETE promoted the phosphorylation of IKBa, which was attenuated by the ILK siRNA. These results indicate that 12-HETE facilitates the activation and nuclear translocation of NF-κB via ILK in ovarian cancer cells.
In addition, to avoid the nonspecific suppression of the ILK siRNA, we then utilized another independent siRNA of ILK (siILK#2) and repeated some important experiments. The knockdown efficiency of siILK#2 was confirmed by Western blot (Figure S3A). We found that the inhibitory effects of 1 µM 12-HETE on the decrease of cell viability and the increase of caspase-3 activity induced by SD were mitigated by siILK#2 (Figure S3B and C). Moreover, 12-HETE repressed the expression of Bax and increased the phosphorylation of NF-κB p65, but these effects were abolished after repressing the expression of ILK with siILK#2 (Figure S3D and E).

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The inhibitory effects of 12-HETE on cell apoptosis are weakened by the inhibition of NF-κB [1]
To illustrate the critical role of NF-κB in the inhibition of cell apoptosis by 12-HETE, we used 5 µM Bay-117082 to block the NF-κB pathway in ovarian cancer cells.17 As shown in Figure 5A, the cell viability increase caused by 1 µM 12-HETE was antagonized by the NF-κB inhibitor. In addition, the inhibitory effects of 12-HETE on SD-induced activation of caspase-3 and caspase-9 were weakened by the inhibition of NF-κB (Figure 5B and C). Moreover, the 12-HETE-mediated decrease of the protein level of Bax was mitigated by Bay-117082 (Figure 5D). These results indicate that 12-HETE protects against cell apoptosis through the ILK/NF-κB pathway in ovarian cancer.

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12-HETE inhibited cell growth and apoptosis via ILK and NF-κB in SKOV3 ovarian cancer cells [1]
To determine whether the anti-apoptotic effect of 12-HETE is specific to OVCAR-3 cells, we examined its roles in SKOV3 ovarian cancer cells. Our results showed that 1 µM 12-HETE inhibited the decrease in cell viability induced by SD and that this effect was attenuated by siILK (Figure 6A). The activation of caspase-3 and increased protein level of Bax were repressed in SKOV3 cells by treatment with 1 µM 12-HETE, while the inhibitory effects of 12-HETE on cellular apoptosis were mitigated by knockdown of ILK (Figure 6B and C). Moreover, 1 µM 12-HETE inhibited the decrease in cell viability, and the 12-HETE-mediated increase in caspase-3 activity and Bax expression was impeded by an inhibitor of the NF-κB pathway (Figure 6D–F). All of the above results imply that 12-HETE promotes cell survival by regulating the ILK/NF-κB pathway in ovarian cancer cells.

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In the AMPA-R-mediated toxicity model, calcium can enter neurons through both AMPA-Rs and VSCCs (Colwell and Levine, 1999; Leski et al., 1999). To examine whether 12-HETE protection is mediated by such depolarization-sensitive channels, toxicity studies were conducted in the presence of 250 nm calcicludine, a toxin that inactivates L-, N-, and P-type calcium channels (Schweitz et al., 1994). Calcicludine treatment reduced the overall level of LDH released after glutamate stimulation (Fig.6A) but also completely prevented 12-(S)HETE protection, suggesting that 12-HETE and calcicludine may share a site of action. To confirm this result and further characterize the population(s) of calcium channel(s) that are sensitive to 12-HETE inhibition, the study was repeated with channel-specific inhibitors. In the presence of the N-type blocker conotoxin GVIa (500 nm, Fig.6B) or the synthetic L-type channel blocker nifedipine (2 μm, Fig. 6C), the results achieved were similar to those observed with calcicludine (i.e., the VSCC blockers reduced toxicity and 12-HETE did not provide additional protection). In contrast, although the P/Q-type calcium-channel toxin agatoxin IVa (250 nm) reduced glutamate toxicity, its protective effect was additive with that of 12-HETE (Fig. 6D), suggesting that agatoxin and 12-HETE affect different targets. These data strongly suggest that 12-HETE prevents glutamate-induced excitotoxicity by inhibiting N- and L-type VSCCs but does not affect P-type channel function. [3]

12-HETE was also shown to increase bovine platelet aggregation induced by thrombin and this effect inhibits prostaglandin E1-induced elevation of intracellular cAMP levels. The capacity of 12-HETE to increase thrombin-induced platelet aggregation was confirmed in human subjects and it was related to [Ca2+] mobilization. To better understand the influence of thrombin on 12-HETE release, Holinstat and colleagues characterized and compared the effects of the activation of thrombin receptors, protease-activated receptor (PAR)1 and 4, on PLA2 activity. The authors showed that in platelets two different PAR-induced signaling pathways provide AA for COX or LOX enzymes. These authors explored the kinetics of 12-HETE formation after activation of WP with either thrombin or PAR1 and 4 activating peptides (AP): formation of 12-HETE is significantly delayed compared with that of TXB2. In particular, PAR1-AP induces an immediate formation of 12-HETE, whereas the formation of this eicosanoid is delayed after exposure of platelets to PAR4-AP. In addition, while PAR1-induced TXB2 formation reaches maximal levels within 15 s, 12-HETE biosynthesis continues for longer time, reaching the plateau only at 120 s. These data are conceivable with the concept that COX-1 is a suicide enzyme, whereas 12-LOX continues to oxidize AA over time and its activity may be considered as a function of the availability of the substrate AA.
The ability of thrombin to increase 12-HETE levels was also confirmed in a recent study conducted by Burzaco and colleagues who further demonstrated the capacity of thrombin to increase 12-HETE biosynthesis in human WP. In this study the role of extracellular ATP in the inhibition of thrombin induced-platelet aggregation, in a dose-dependent manner, and also in the inhibition of inositol phosphates generation and intracellular [Ca2+] mobilization has been evidenced.
Concerning platelet activation induced by collagen, Sekiya showed that exogenous 12-HETE, added to bovine platelets, inhibits the release of AA from phospholipids and collagen-induced platelet aggregation, in agreement with a previous study of Takenaga [2].

Measurement of single neuron, cytosolic calcium concentrations ([Ca2+]i).[3]
Neurons were seeded onto glass coverslips at a density of 1 × 106/cm2. After 8 d in culture, the cells were washed twice in a HEPES-buffered Krebs–Ringer saline (KRS) containing (in mm): 125 NaCl, 5 KCl, 1 Na2HPO4, 1 MgSO4, 1 CaCl2, 5.5 glucose, and 20 HEPES, pH 7.2. Coverslips were then placed in a 4 μm solution of fura-2 AM in KRS for 22 min at room temperature with continuous gentle agitation. Next, cells were washed and incubated for an additional 22 min in fresh KRS (Grimaldi and Cavallaro, 1999). The coverslips were then mounted in a chamber and perfused with KRS containing 0.002% methylcellulose (vehicle) at a flow rate of ∼800 μl/min. Images were acquired by an inverted microscope equipped with a 40× neofluar lens and an intensified CCD camera attached to a desktop computer. Metafluor software (Universal Imaging Corporation, West Chester, PA) was used to analyze experimental data. Ratio values, obtained by alternately exciting the preparations at wavelengths of 340 and 380 nm and recording the emitted light at 510 nm, were converted into calcium concentrations using the Grynkiewicz equation (subtracted equation form) (Grynkiewicz et al., 1985). Calibration values for this equation (Rmax − R,R − Rmin) were acquired by exposing neurons either to 10 μmionomycin in KRS containing 10 mm calcium or to 20 mm EGTA in calcium-free KRS, respectively.

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Intracellular cAMP measurements. [3]
Intracellular cAMP levels were measured using a commercially available enzyme immunoassay kit. Before assay, cortical neuron cultures were exposed to 100 μm isobutylmethylxanthine and varying concentrations of 12-HETE for 10 min before and for 15 min during exposure to 5 μm forskolin. After this time cells were lysed with a detergent solution provided by the assay manufacturer and intracellular cAMP concentrations were assessed. Assays were conducted according to the manufacturers' instructions.

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Agonist-stimulated [35S] GTPγS binding studies. [3]
Rat forebrains (minus the cerebellum) were disrupted on ice in a buffer containing 0.32m sucrose, 50 mm Tris–HCl, pH 7.4, 3 mm MgCl2, and 1 mm EGTA, pH 7.4. The homogenates were centrifuged for 10 min at 1000 × g, the pellets were discarded, and the supernatants were then recentrifuged for 30 min at 3000 ×g. The resulting pellet was triturated in ice-cold Tris buffer containing (in mm): 50 Tris–HCl, pH 7.4, 3 MgCl2, and 1 EGTA, pH 7.4; it was then recentrifuged for 30 min at 3000 × g. The pellet was then suspended in 10 vol of Tris buffer containing 0.03% digitonin, incubated at room temperature for 15 min, and centrifuged for 30 min at 3000 × g. Membranes were then dispersed in Tris buffer; the protein concentration was assayed using Bradford's reagent and the membranes were then frozen in aliquots at −80°C. On the day of the assay, an aliquot was thawed and diluted in assay buffer containing (in mm): 20 HEPES, 100 NaCl, 3 MgCl2, and 0.5 EGTA, as well as 0.1% (w/v) BSA, pH 7.4. The aliquot was then centrifuged for 30 min at 3000 × g. Membranes were subaliquoted into 1 ml of assay buffer containing 15 μg of protein, 30 μm GDP, 160 pm[35S]GTPγS, and varying concentrations of 12-(S)HETE/12-HETE. Nonspecific binding was determined in the presence of 30 μm “cold” GTPγS. Samples were incubated for 1 hr at 23°C, after which time the reaction was terminated by rapid filtration under vacuum through Whatman GF/B glass fiber filters (Whatman, Maidstone, UK), followed by four washes with cold assay buffer. Radioactivity bound to the filters was determined by liquid scintillation counting. The data were plotted and EC50 values were calculated using the Prism software.

Cell Viability Assay[1]
Cell Types: Ovarian cancer OVCAR-3 and SKOV3 cells
Tested Concentrations: 0, 0.2, 0.5, and 1 µM
Incubation Duration: 0, 24, 48, 72, and 96 hrs (hours)
Experimental Results: Inhibited the decrease in cell viability induced by SD in a dose-dependent manner. 1 µM 112-HETE treatment Dramatically mitigated the decrease in cell viability under conditions of SD.

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Western Blot Analysis[1]
Cell Types: Ovarian cancer OVCAR-3 and SKOV3 cells
Tested Concentrations: 1 µM
Incubation Duration:
Experimental Results: Led to increased levels of NF-κB p65 phosphorylation. Caused a significant increase in the protein levels of nuclear NF-κB p65, which was accompanied by diminished levels of NF-κB p65 in the cytoplasm.

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MTT assay [1]
Cells (OVCAR-3 and SKOV3) were cultured in 96-well plates at a density of approximately 1×104 cells per well. After culturing in 10% FBS DMEM medium for 8–12 hours, the cells were cultured in DMEM without serum overnight and treated with the indicated reagents by group in low-glucose DMEM without FBS (serum deprivation [SD]). Solvent control and other agents were added at the indicated concentrations every 24 hours. After being treated as different groups for 48 hours, the cells were incubated in 0.5% MTT, which is a yellow mitochondrial dye and is dissolved in sterile PBS buffer, for 4 hours at 37°C, and then, the reaction was terminated by incubating the cells with DMSO for 10 minutes. The spectrophotometer absorbance at 540 nm was measured. The amount of blue formazan dye formed from MTT is proportional to the number of surviving cells.

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LDH assay [1]
The activity of LDH was used to detect the levels of LDH, which was released into the culture media, and was measured by a cytotoxicity detection kit from Beyotime Institute of Biotechnology. The proportion of injured cells in the cultures was determined by comparing the LDH activity of the medium with the LDH activity after complete cell lysis or total LDH activity. The maximum LDH activity was determined using medium containing Triton-lysed cell supernatants. The experiments were carried out according to the manufacturers’ instructions. A portion of the culture medium was treated with an equal volume of LDH substrate solution for 30 minutes and then stopped with 5 volumes of 0.1 M NaOH; a spectrophotometer was used to measure the absorbance at 440 nm in sister cultures that were treated with 1/100 volume of 10% Triton X-100 and incubated for 30 minutes at 37°C.

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Caspase-3 and caspase-9 activity assays [1]
We measured the cleavage of chromogenic caspase substrates, Ac-DEVD-pNA (acetyl-Asp-Glu-Val-Asp p-nitroanilide), a caspase-3 substrate, and Ac-LEHD-pNA (acetyl-Leu-Glu-His-Asp p-nitroanilide), a caspase-9 substrate, to calculate caspase-3 and caspase-9 activities, respectively. The experiment was performed according to the manufacturer’s protocols. Approximately 50 µg of total protein was added to the reaction buffer containing Ac-DEVD-pNA (2 mM) or Ac-LEHD-pNA (2 mM) and then incubated at 37°C for 2 hours. The absorbance of yellow pNA cleaved from its corresponding precursors was measured using a spectrometer at 405 nm. The specific caspase activities, normalized to the total protein of the cell lysates, were then expressed as the fold change relative to the baseline of control cells cultured in DMEM with 10% FBS.

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Western blot assay [1]
We used protein lysis buffer together with the appropriate concentrations of proteinase inhibitors and phosphatase inhibitors to extract cell proteins. Then, the Bradford assay was used to detect the total protein concentration. The protein samples were fractionated by SDS-PAGE (12% polyacrylamide gels) followed by transfer onto nitrocellulose membranes. After incubation in blocking buffer (20 mM Tris, pH 7.6, 150 mM NaCl, and 0.1% Tween-20) containing 5% nonfat dry milk powder, the membranes were incubated with the indicated antibodies overnight at 4°C, followed by reaction with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. Enhanced chemiluminescent reagents were used to visualize the protein bands by chemiluminescence detection kit. Beta-actin was used as an internal control.

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Solution preparation. [3]
Solutions of HETEs such as 12-HETE and other lipophilic compounds were prepared by evaporating a 10 mm ethanolic solution (under a stream of nitrogen) in a siliconized microcentrifuge tube. Dimethyl sulfoxide (<0.05% of final volume) was added to ethanol to prevent the lipophil from completely drying onto the tube wall. After evaporation, 1 ml of culture medium was added and the drug was dispersed using a high-power sonic probe. Special attention ensured that the solutions did not generate foam or become hot to the touch. After dispersal, all solutions were brought to their final volume in siliconized glass tubes by mixing with an appropriate quantity of culture medium.

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Neuronal cultures. [3]
Primary cortical neuron cultures were prepared according to a previously published method (Grimaldi and Cavallaro, 1999). Briefly, fetuses were extracted from Wistar rats that had been pregnant for 17 d. The cortices were then dissected out, cut into small pieces, and incubated with papain (1 mg/ml) for 9 min at 37°C. After this time the tissue was dissociated by passage through a fire-polished Pasteur pipette, and the resultant cell suspension was separated by centrifugation over a gradient consisting of 10 mg/ml bovine serum albumin (BSA) and 10 mg/ml ovomucoid (a trypsin inhibitor) in Earle's balanced salt solution. The pellet was then resuspended in high-glucose, phenol red-free DMEM containing 10% fetal bovine serum, 2 mm glutamine, 100 IU of penicillin, and 100 μg/ml streptomycin. Cells were counted and tested for viability using the trypan blue exclusion test; next, 3.2 × 105 cells/well were seeded onto the inner 24 wells of a poly-d-lysine-coated 48 well plate. The outer wells of the plate were filled with water to reduce evaporation from the cultures. Ninety-six hours after seeding, a cocktail containing 10 μm fluorodeoxyuridine and 10 μm uridine was added to block the growth of glial cells. After 7 d, 100 μl of fresh medium containing the glial-cell-inhibiting cocktail was added again to compensate for nutrient depletion.

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NMDA-R-mediated toxicity procedure. [3]
Cortical neurons were cultured for 13–15 d in vitro. Before the experiment, half of the medium in which the cells were maintained was removed and kept for later use. Glutamate toxicity was examined by exposing the cultures to 200 μm glutamate for 15 min in a magnesium-free saline solution composed of (in mm): 125 NaCl, 25 glucose, 10 HEPES, pH 7.4, 5 KCl, and 1.8 calcium chloride, as well as 2.5% fatty-acid-free BSA. After exposure, cells were washed twice with saline and incubated in a medium composed of 70% original culture medium (in which the neurons had been cultured for the past 13–15 d) and 30% fresh medium. The cells were then incubated for 18 hr, after which time lactate dehydrogenase (LDH) levels in the media were examined and used as an index of cell toxicity. Preliminary studies demonstrated that glutamate toxicity was prevented by 500 nm MK-801, confirming an NMDA-R-mediated mechanism (data not shown).

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AMPA- and kainate receptor-mediated toxicity procedures. [3]
To examine AMPA- and kainate receptor-mediated toxicity, neurons were cultured for 6–9 d and then exposed to 100 μmglutamate and 25 μm cyclothiazide (used to prevent AMPA-R desensitization) and 500 nm MK-801 for 18–20 hr before analysis.
The neuron preparation technique described above results in a primarily neuronal culture, although a limited number of astrocytes remain. This cell type is resistant to glutamate toxicity (Amin and Pearce, 1997), although such cell death has been reported in the presence of cyclothiazide (David et al., 1996). Initial studies were performed to confirm that astrocytes did not significantly contribute to AMPA–kainate toxicity in our cultures. When astrocytes were exposed to glutamate under the same conditions used on neuron preparations and left for 20 hr, LDH levels increased to only 105% of background, compared with 150–200% in neuron-enriched cultures (data not shown). Therefore, it was concluded that astrocyte contamination did not substantially contribute to the effects of glutamate in our neuronal cultures.

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Toxicity assay. [3]
Cell toxicity was assessed 18–20 hr after insult by measuring LDH release into the phenol red-free culture media according to the method of Decker and Lohmann-Matthes (1988). Experiments were conducted with quadruplicate values at each point, and all plates contained glutamate (positive) and nonglutamate (negative) controls. The required assay development time varied between culture batches and number of days in vitro, and values were recorded when the positive control wells demonstrated absorbance values at 495–650 nm of 0.3–0.4 au. The assay was validated by comparison with a mitochondrial function viability assay [2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide (XTT) assay; Roehm et al., 1991]. The results obtained with the two systems were similar, although LDH release was used in this study because it provided a greater signal-to-noise ratio than the XTT assay. The ratio of mean LDH levels in negative and positive controls was used as an index of culture quality. Data from trials in which the mean LDH level in the negative controls exceeded 60% of the maximum (positive control) values were not included.

Metabolism / Metabolites
12-HETE is a known human metabolite of arachidonic acid.

Pertussis toxin studies [3]
Pertussis toxin (PTx, an agent that inactivates Gi/o proteins) was used to investigate whether 12-HETE exerts its effect by direct action on N- and L-type VSCCs or by activating a Gi/o-protein-coupled receptor (Zamponi and Snutch, 1998; Kaneko et al., 1999). After treatment with 100 ng/ml PTx overnight, the anti-excitotoxic effect of 12-(S)HETE was eliminated (Fig.7), which suggests that 12-(S)HETE inhibits N- and L-type VSCCs via Gi/o-protein linkage rather than through a direct action. The effect of PTx on 12-(S)HETE-inhibited calcium influx was also examined by calcium imaging. As with the glutamate toxicity studies, PTx treatment eliminated the inhibitory effect of 12-HETE on glutamate-induced [Ca2+]i (Fig.8A,C), which suggests that 12-(S)HETE inhibits glutamate-stimulated calcium influx via a Gi/o-protein-coupled mechanism.

[1]. 12-HETE facilitates cell survival by activating the integrin-linked kinase/NF-κB pathway in ovarian cancer. Cancer Manag Res. 2018 Nov 16;10:5825-5838.

[2]. Analysis, physiological and clinical significance of 12-HETE: a neglected platelet-derived 12-lipoxygenase product. J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Aug 1;964:26-40.

[3]. 12-hydroxyeicosatetrenoate (12-HETE) attenuates AMPA receptor-mediated neurotoxicity: evidence for a G-protein-coupled HETE receptor. J Neurosci. 2002 Jan 1;22(1):257-64.

(5Z,8Z,10E,14Z)-12-hydroxyicosatetraenoic acid is the (5Z,8Z,10E,14Z)-stereoisomer of 12-HETE. It has a role as a mouse metabolite. It is a conjugate acid of a 12-HETE(1-).
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid is a metabolite generated from arachidonic acid by 12S-type arachidonate 12-lipoxygenase in platelets. 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE) may be involved in inflammation and pruritus.
A lipoxygenase metabolite of ARACHIDONIC ACID. It is a highly selective ligand used to label mu-opioid receptors in both membranes and tissue sections. The 12-S-HETE analog has been reported to augment tumor cell metastatic potential through activation of protein kinase C. (J Pharmacol Exp Ther 1995; 274(3):1545-51; J Natl Cancer Inst 1994; 86(15):1145-51)

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Background: The dysfunction of cell apoptosis is an important event in the progression of cancer, and the growth of cancer cells is negatively regulated by cell apoptosis. In different types of cancers, inhibition of cellular apoptosis is often observed in the cancerous tissue, and increased resistance to apoptosis is a hallmark of cancer. Although previous studies have shown that 12-lipoxygenase (12-LOX)/12-hydroxyeicosatetraenoic acid (12-HETE) is activated and upregulated in different types of cancers, the consequences of 12-LOX/12-HETE upregulation and its precise roles in the survival of ovarian carcinoma cells are still unknown.
Methods: MTT assays, caspase activity assays, lactate dehydrogenase (LDH) assays, and Western blot analysis were the methods used in this study.
Our study suggests that 12-HETE inhibits the mitochondria-dependent apoptosis pathway through the ILK pathway and that NF-κB acts as a downstream target of 12-HETE-activated ILK. This study implies that 12-LOX/12-HETE and its downstream targets (ILK and NF-κB) play critical roles in promoting the survival of ovarian cancer cells and positively regulate the progression of ovarian cancer, which indicates a new potential target for future treatment of ovarian cancer. [1]

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This review reports the up to now knowledge about 12-HETE, in particular focusing on the role of the biosynthesis of this 12-LOX metabolite in platelets. The analysis of the literature reveals the presence of different analytical methods for the detection of eicosanoids, with good specificity, but the sample preparation still needs to be implemented. The majority of analytical techniques present in the literature are independent from the source of this AA metabolite, even if different cell types may differ in the kinetic of 12-HETE production. For this reason, who intends to actually study platelet 12-HETE from a physiological and clinical point of view cannot avoid taking into consideration the matrix in which the measurements should be performed. We think that serum might be the biological sample ideally suitable to this goal for several reasons. The handling of serum is simple, convenient and time saving because no steps are required for cell isolation. In the serum prostanoids produced during clotting are the result of the interaction between various cell types, simulating in vivo conditions. Even if this type of sample do not reflect basal concentrations, it constitutes a suitable and easy-to-obtain ex vivo system to investigate simultaneously the activity of both platelet COX and LOX enzymes.

However, we believe that essential premise is to study in depth the kinetic of 12-HETE formation in platelets because some authors reported a continuous production of this molecule. The amount of 12-HETE produced from rat platelets aggregated by collagen increased continuously, even after 115 min as platelet 12-LOX is able to utilize, not only AA endogenously present in the cell, but also that one derived from plasma phospholipids. Even more important, this enzyme is resistant to inactivation. These data has been confirmed also by Sautebin in humans. So all these points must be taken into account in order to develop an analytical method for measurement of 12-HETE derived from platelets.
As documented, the information on the physiological and pathological functions of this 12-LOX metabolite are so far not completely clarified. Conflicting results have been reported about the role of 12-HETE in platelet function. Indeed, whatever the agonist used to induce platelet aggregation, 12-HETE showed to exert both platelet pro- and anti-aggregant activity, with the exception of platelet aggregation induced by thrombin, in which exogenously added 12-HETE was shown to have proaggregatory activity.

The increased platelet proaggregatory activity of 12-HETE may be of relevance in controlling the occurrence of thrombotic phenomena in human. In this context the observation that 12-HETE synthesis is involved in transient heparin-induced platelet activation during carotid endarterectomy is of particular relevance. So the understanding of mechanisms by which 12-LOX and its metabolites modulate platelet function might be of help not only in elucidating the role of this 12-hydroxy fatty acid in the thrombotic process, but also may provide the rationale for the development of a novel class of anti-platelet drugs. [2]

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While the importance of cyclooxygenase (COX) in platelet function has been amply elucidated, the identification of the role of 12-lipoxygenase (12-LOX) and of its stable metabolite, 12-hydroxyeicosatretraenoic acid (12-HETE), has not been clarified as yet. Many studies have analysed the implications of 12-LOX products in different pathological disorders but the information obtained from these works is controversial. Several analytical methods have been developed over the years to simultaneously detect eicosanoids, and specifically 12-HETE, in different biological matrices, essentially enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), high performance liquid chromatography (HPLC) and mass spectrometry coupled with both gas and liquid chromatography methods (GC- and LC-MS). This review is aimed at summarizing the up to now known physiological and clinical features of 12-HETE together with the analytical methods used for its determination, focusing on the critical issues regarding its measurement. [2]

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12-hydroxyeicosatetraenoic acid (12-HETE) is a neuromodulator that is synthesized during ischemia. Its neuronal effects include attenuation of calcium influx and glutamate release as well as inhibition of AMPA receptor (AMPA-R) activation. Because 12-HETE reduces ischemic injury in the heart, we examined whether it can also reduce neuronal excitotoxicity. When treated with 12-(S)HETE, cortical neuron cultures subjected to AMPA-R-mediated glutamate toxicity suffered up to 40% less damage than untreated cultures. The protective effect of 12-(S)HETE was concentration-dependent (EC50 = 88 nm) and stereostructurally selective. Maximal protection was conferred by 300 nm 12-(S)HETE; 300 nm 15-(S)HETE was similarly protective, but 300 nm 5-(S)HETE was less effective. The chiral isomer 12-(R)HETE offered no protection; neither did arachidonic acid or 12-(S)hydroperoxyeicosatetraenoic acid. Excitotoxicity was calcium-dependent, and 12-(S)HETE was demonstrated to protect by inactivating N and L (but not P) calcium channels via a pertussis toxin-sensitive mechanism. Calcium imaging demonstrated that 12-(S)HETE also attenuates glutamate-induced calcium influx into neurons via a pertussis toxin-sensitive mechanism, suggesting that it acts via a G-protein-coupled receptor. In addition, 12-(S)HETE stimulates GTPgammaS binding (indicating G-protein activation) and inhibits adenylate cyclase in forskolin-stimulated cultures over the same concentration range as it exerts its anti-excitotoxic and calcium-influx attenuating effects. These studies demonstrate that 12-(S)HETE can protect neurons from excitotoxicity by activating a G(i/o)-protein-coupled receptor, which limits calcium influx through voltage-gated channels.[3]

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During the drafting of this manuscript a report was published indicating that 12-(S)HETE has some affinity for the low-affinity leukotriene B4 receptor (BLT2) (Yokomizo et al., 2001). However, the features of BLT2 are considerably different from those described here; in particular, this receptor is not pertussis-toxin sensitive, 12-(S)HETE binds to BLT2 with an EC50 in the micromolar rather than nanomolar range, and most importantly, mRNA to BLT2 is found in almost every tissue except the brain (Yokomizo et al., 2000).

In summary, the data presented in this study demonstrate that 12-(S)HETE significantly attenuates neuronal AMPA toxicity by reducing calcium influx through N- and L-type calcium channels during glutamate exposure. The protective effect of 12-HETE is stereostructurally defined and is mediated via a PTx-sensitive Gi/o protein linkage. As with other Gi/o-protein-coupled receptor systems, the putative 12-(S)HETE receptor inhibits adenylate cyclase activity in addition to inhibiting VSCCs. The concentration range over which 12-(S)HETE inhibits cAMP formation and stimulates GTPγS binding is similar to that over which it reduces glutamate toxicity. Together, these data strongly support the existence of a G-protein-coupled 12-(S)HETE receptor in rat cortical neuronal membranes, which may act as a neuroprotective system during excitotoxic events such as ischemia.[3]

C20H32O3

320.46628

320.235

C, 74.96; H, 10.07; O, 14.98

71030-37-0

12-HETE-d8;2525175-25-9

13786989

Colorless to light yellow liquid

1.0±0.1 g/cm3

487.7±45.0 °C at 760 mmHg

262.8±25.2 °C

0.0±2.8 mmHg at 25°C

1.514

5.45

2

3

14

23

392

0

CCCCC/C=C\CC(/C=C/C=C\C/C=C\CCCC(=O)O)O

ZNHVWPKMFKADKW-VXBMJZGYSA-N

InChI=1S/C20H32O3/c1-2-3-4-5-10-13-16-19(21)17-14-11-8-6-7-9-12-15-18-20(22)23/h7-11,13-14,17,19,21H,2-6,12,15-16,18H2,1H3,(H,22,23)/b9-7-,11-8-,13-10-,17-14+

(5Z,8Z,10E,14Z)-12-hydroxyicosa-5,8,10,14-tetraenoic acid

71030-37-0; 12-Hete; 5,8,10,14-Eicosatetraenoicacid, 12-hydroxy-, (5Z,8Z,10E,14Z)-; 12-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid; (5Z,8Z,10E,14Z)-12-hydroxyicosatetraenoic acid; (+/-)12-HETE; 12-Hydroxyeicosatetraenoic acid; (5Z,8Z,10E,14Z)-12-hydroxyicosa-5,8,10,14-tetraenoic acid;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)