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CELLULAR AND MOLECULAR
Shanghai Institute of Materia Medica, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
Received March 18, 2005; accepted May 26, 2005.
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Abstract |
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; Spaargaren et al., 1991). Following ligand binding, EGFRs oligomerize, become autophosphorylated, and finally are activated (Emlet et al., 1997). EGFR activates two major downstream pathways: phosphatidylinositol 3-kinase (PI3K)/3-phosphoinositide-dependent protein kinase 1 (PDK-1)/Akt (also known as protein kinase B) pathway and Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) signaling pathway (Carpenter and Cohen, 1990; Emlet et al., 1997; Okano et al., 2000). The PI3K/Akt pathway plays important roles in a number of cellular events like diabetes, cell proliferation, differentiation, transformation, and apoptosis (Dumont et al., 1989; Lou et al., 2002). Aberrant activation of the PI3K/Akt pathway has been widely implicated in many cancers, and drugs against this pathway are likely to have broad applications for treating different types of cancer (Luo et al., 2003).
Phospholipase A2 (PLA2) enzymes represent a family of esterases catalyzing the hydrolysis of the sn-2 position of membrane glycerophospholipids to liberate free fatty acids and lysophospholipids (Kudo and Murakami, 2002). There are four major families of PLA2s: the 14-kDa secretory PLA2 (sPLA2), the Ca2+-independent PLA2 (iPLA2), the 85-kDa cytosolic PLA2 (cPLA2), and platelet-activating factor acetyl-hydrolases (Murakami et al., 2000; Kudo and Murakami, 2002). To study the role of different PLA2s in eicosanoid production as well as other pathophysiological functions such as inflammation, considerable effort has been put into developing specific inhibitors for different PLA2s. These inhibitors included marine natural products and PLA2 substrate and product analogs (Hope et al., 1993). However, many of the PLA2 inhibitors originally thought to be selective for a specific PLA2 are now known to have multiple other effects (Cummings et al., 2000).
12-Epi-scalaradial (SLD), a natural product isolated from marine sponge (Cacospongia species), has been shown to be a selective inhibitor of sPLA2 (De Carvalho and Jacobs, 1991). SLD efficiently inhibited bee venom PLA2 (IC50 = 0.07 µM) and purified human recombinant type II sPLA2 activity in vitro (IC50 = 5.4 µM) but displayed weak inhibition of cytosol 85-kDa PLA2 from U937 cells (IC50 = 28 µM) (De Carvalho and Jacobs, 1991; Hope et al., 1993). However, whether this compound has other effects besides inhibiting sPLA2 activity is still unknown. Previous studies demonstrated that SLD exerted inhibitory effect on HL-60 cell proliferation (Liu and Levy, 1997). Recent unpublished work in our laboratory indicated that SLD also inhibited the proliferation of many other cell lines. Given that EGFR/PI3K/Akt signaling pathway plays important roles in cell proliferation, we investigated the effect of SLD on EGFR-mediated signaling. Our results revealed that SLD inhibited EGFR-mediated Akt phosphorylation, and this novel effect was independent of sPLA2.
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Materials and Methods |
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-Phosphatidylinositol was purchased from Avanti Polar Lipids (Alabaster, AL). [
-32P] ATP and protein G-Sepharose were purchased from Amersham Biosciences AB (Uppsala, Sweden). FuGENE 6 transfection reagent was purchased from Roche Diagnostics (Mannheim, Germany). pEGFP plasmid and pEGFP-PDK-1 plasmid were kind gifts from Dr. Jongkyeong Chung (Korea Advanced Institute of Science and Technology, Taejon, Korea). Human group IIA and X sPLA2 plasmids were kind gifts from Dr. Gerard Lambeau (Institut de Pharmacologie Moleculaire et Cellulaire, Valbonne, France) (Cupillard et al., 1997). Antibodies to Akt, ERK1/2, phospho-Akt (Ser473), and phospho-ERK1/2 were purchased from Cell Signaling (Beverly, MA). Antibody to PI3 kinase p85 was purchased from Upstate Biotechnology (Lake Placid, NY). Cell Culture and Transfection. Hepatocellular carcinoma BEL-7402 cells were from Cell Bank of Shanghai Institute for Biological Sciences, Chinese Academy of Science (Shanghai, China). Breast cancer MDA-MB-435 cells were from American Type Culture Collection (Manassas, VA). Human dermal microvascular endothelial cells (HMECs) were from Cambrex Bio Science Walkersville (Walkersville, MD). BEL-7402 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37°C in an atmosphere of 5% CO2. MDA-MB-435 cells and HMECs were maintained in Dulbecco's modified Eagle's medium containing 10% FBS at 37°C with 5% CO2. All the cells were plated in six-well plates and starved in serum-free medium for 16 h before drug treatment. For transient transfection, cells grown in six-well plates at 70% confluence were transfected with pEGFP, pEGFP-PDK-1, human group IIA, or human group X sPLA2 plasmids using the FuGENE 6 transfection reagent according to the manufacturer's instructions. After transfection for 24 h, cells were starved in serum-free RPMI 1640 medium for 16 h before drug treatment.
Preparation of Cell Lysates and Western Blot. After drug treatment, cells were washed twice with ice-cold PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4), and total cell lysates were collected in SDS sample buffer (50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromphenol blue, and 10% glycerol). Cell lysates containing equal amount of protein were separated by SDS-PAGE and transferred to polyvinylidine difluoride membranes. After being blocked in 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20, pH 7.6, membranes were incubated with the appropriate primary antibodies at 4°C overnight and exposed to secondary antibodies for 2 h at room temperature. Immunoreactive proteins were visualized using the enhanced chemiluminescence system from Pierce Chemical (Rockford, IL).
PDK-1 Translocation Assay. BEL-7402 cells were grown on glass coverslips in six-well plates and transfected with pEGFP or pEGFP-PDK-1 plasmids. After transfection for 24 h, cells were starved in serum-free RPMI 1640 medium for 16 h and then stimulated with EGF in the presence or absence of SLD or wortmannin. Stimulation was terminated by washing cells with ice-cold PBS and fixing cells with immediate addition of 4% paraformaldehyde in PBS for 10 min at 4°C. Fixed cells were mounted on slide glasses with 50% glycerol in carbonate buffer (pH 9.0) and observed with a laser-scanning confocal microscope (Leica, Wetzlar, Germany).
PI3K Activity Assay. The assay was performed as described (Whitman et al., 1985). Briefly, cells were washed twice with ice-cold PBS and lysed on ice in lysis buffer [20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10% (v/v) glycerol, 1% Nonidet P-40, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 50 mM sodium fluoride, 2 µM leupeptinin, 2 µM aprotinin, and 1 mM phenylmethylsulfonyl fluoride]. Insoluble material was removed by centrifugation at 12,000 rpm at 4°C for 10 min. Cell lysates (500 µg) were subjected to immunoprecipitation with anti-PI3K p85 antibody for 2 h followed by the addition of 20 µl of 1:1 slurry of protein G-Sepharose beads for an additional hour at 4°C. The immunocomplexes were incubated with 0.1 mg/ml L--phosphatidylinositol, 50 µM ice-cold ATP, 15 µCi [-32P] ATP, and 20 mM MgCl2 for 30 min at 30°C in a final volume of 80 µl. Twenty microliters of 8 M HCl was added to terminate the incubation. The lipids were extracted with 200 µl of chloroform/methanol (1:1) and centrifuged (300g) for 15 min at 4°C. The lower organic phases were resolved on a 1% potassium oxalate-pretreated silica gel 60-Å thin-layer chromatography plate (Merck, Darmstadt, Germany) by chloroform/acetone/methanol/acetic acid/water (46:17:15:14:8, v/v) and visualized by autoradiography. Identified spots containing radioactive phosphatidylinositol-3-phosphate were scraped off the silica gel plates and quantitated by scintillation counting.
RT-PCR. BEL-7402 cells were transfected with human group IIA or X sPLA2 plasmids. After transfection for 24 h, cells were starved in serum-free RPMI 1640 medium for 16 h. Total RNA was isolated using TRIzol reagent (Sangon, Shanghai, China) following the manufacturer's protocols. cDNA mixtures were prepared in a 20-µl reaction using an RT-PCR kit (Promega). Briefly, 2 µg of total RNA was reverse-transcribed using 200 units of Moloney murine leukemia virus RTase H in the presence of 0.5 µg oligo(dT)18, 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 0.5 mM of each dNTP, and 20 units of RNase inhibitor. The reaction mixture was incubated for 60 min at 42°C (transcription) followed by 15 min at 70°C (inactivation of reverse transcription). PCR was performed in 50-µl reactions containing 10 mM Tris-HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 0.2 mM of each dNTP, 5 units of Taq polymerase, and 1 µM of the specific primers. The group IB sPLA2 primer sequences used were 5'-TCC TTG TGC TAG CTG TGC TG-3' and 5'-TGA AGG CCT CAC ACT CTT TG-3' (Degousee et al., 2002). The group IIA sPLA2 primer sequences were 5'-ATG AAG ACC CTC CTA CTG TT-3' and 5'-TCA GCA ACG AGG GGT GCT CC-3' (Degousee et al., 2002). The group IIA sPLA2 primer sequences were 5'-TTG GTT CCT GGC TTG TAG TGT G-3' and 5'-TGG GTT GTA GCT CCG TAG GTT T-3' (Degousee et al., 2002). The group V sPLA2 primer sequences were 5'-GAT CCT GGA ACT GGC AGG AA-3' and 5'-TCA GTC ACA CTT GGG CGA GT-3' (Degousee et al., 2002), and the group IV cPLA2 primer sequences were 5'-CTC TAG TCC TCC GTT CAA GGA AC-3' and 5'-TTG CAA ACT GCC TCA GCA TCA-3' (Wu et al., 1998). The PCR reaction for group IB, IIA, V, and X sPLA2 was conducted for 4 min at 95°C for 35 cycles of amplification using a step program (95°C, 30 s; 56°C, 45 s; 72°C, 50 s), followed by 10 min at 72°C to ensure a complete extension of the amplified DNA. The PCR reaction for group IV cPLA2 was conducted for 4 min at 95°C for 30 cycles of amplification using a step program (94°C, 45 s; 56°C, 45 s; 72°C, 1.5 min), followed by a 10-min final extension at 72°C. The PCR products were electrophoresed on a 1.5% agarose gel and stained with ethidium bromide.
Measurement of sPLA2 Activity. BEL-7402 cells were transfected with human group IIA or X sPLA2 plasmids. After transfection for 24 h, cells were starved in serum-free RPMI 1640 medium for another 16 h, then collected in PBS (108 cells/ml) containing 0.34 M sucrose, 10 mM HEPES, 100 µg/ml phenylmethylsulfonyl fluoride, 1 mM tosyl-leucyl chloromethylketone, 0.1 mM leupeptin, and 5 mM EDTA (final pH 7.0) (Blom et al., 1998). The suspended cells were sonicated at 21 kHz and 8-µm peak-to-peak amplitude for 3x 15 s at 4°C. sPLA2 activity was measured using sPLA2 Activity Assay Kits from Cayman Chemical.
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Results |
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Next, we investigated the role of SLD in EGFR-mediated Akt activation. We incubated BEL-7402 cells with SLD for 15 min before EGF stimulation and observed SLD dose-dependently inhibited EGF-stimulated Akt phosphorylation with an IC50 of 2.9 ± 0.44 µM (Fig. 1A). In addition, SLD inhibited EGF-stimulated Akt phosphorylation in a treatment schedule-dependent manner. Only pretreatment of cells with SLD before EGF stimulation, but not treatment of cells with SLD plus EGF simultaneously, or treatment of cells with SLD after EGF stimulation, could SLD markedly inhibit Akt phosphorylation (Fig. 1B), suggesting that SLD acts on steps upstream of Akt.
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; Beck-Speier et al., 2003; Bishop-Bailey and Warner, 2003). We incubated BEL-7402 cells with different doses of thioetheramide-PC before EGF stimulation. To our surprise, thioetheramide-PC failed to inhibit Akt phosphorylation even at a dose of 30 µM (Fig. 3A). AACOCF3 (10 µM), a specific inhibitor of cPLA2 (Street et al., 1993), methyl arachidomyl fluorophosphonate (MAFP; 10 µM), an inhibitor of cPLA2 and iPLA2 (Lio et al., 1996; Cummings et al., 2000), and U73122
[GenBank]
(10 µM), an inhibitor of phospholipase C (Zheng et al., 1995), had no effect on EGF-stimulated Akt phosphorylation, either (Fig. 3, B-D). In addition, indomethacin (10 µM), an inhibitor of cyclooxygenases, which are downstream enzymes of PLA2 and convert AA to prostaglandins (Touhey et al., 2002), could not inhibit EGF-stimulated Akt phosphorylation (Fig. 3E). These results indicate the inhibitory effect on EGFR-mediated Akt phosphorylation is specific for SLD.
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SLD Blocked EGF-Induced PDK-1 Membrane Translocation. Akt activation requires phosphorylations at two critical residues: Thr308 at the activation loop of the catalytic domain and Ser473 at the hydrophobic C-terminal domain (Alessi et al., 1996). Upon EGF stimulation, PI3K increases the levels of phosphatidylinositol 3,4,5-trisphosphate, which recruits PDK-1 as well as Akt to the plasma membrane where PDK-1 phosphorylates Thr308 of Akt (Stephens et al., 1998; Scheid et al., 2002). Previous results showed that only using pretreatment protocol was SLD able to inhibit EGF-stimulated Akt phosphorylation. Therefore, we hypothesized that SLD inhibits the upstream regulators of Akt. First, we examined the effect of SLD on PDK-1 membrane translocation. Green fluorescence protein (GFP)-fused PDK-1 plasmid was transfected into BEL-7402 cells, and the effect of SLD on translocation of GFP-PDK-1 protein to plasma membrane was visualized by laser-scanning confocal microscope. As seen from Fig. 4, EGF rapidly induced GFP-PDK-1 membrane translocation within 3 min, and this translocation was totally inhibited by pretreatment with SLD (10 µM) for 15 min. As a positive control, wortmannin also effectively abrogated the PDK-1 membrane translocation. However, localization of GFP was not affected by EGF stimulation (Fig. 4B), indicating that this assay is specific for PDK-1.
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; Kudo and Murakami, 2002). However, our results showed AA (30 µM) could not rescue SLD inhibition of EGF-stimulated Akt phosphorylation (Fig. 6A), suggesting that this inhibitory effect of SLD may be independent of sPLA2. To confirm this hypothesis, we first used ERK1/2 as a control to determine whether AA is active or not in our system because AA has been reported to stimulate ERK1/2 phosphorylation (Dulin et al., 1998; Silfani and Freeman, 2002). As seen from Fig. 6B, direct addition of 30 µM AA to BEL-7402 cells rapidly stimulated ERK1/2 phosphorylation (Fig. 6B). Second, we tested the effect of SLD on ERK1/2 phosphorylation. Although we did not see the inhibitory effect of SLD on EGF-stimulated ERK1/2 phosphorylation (data not shown), we clearly observed treatment of BEL-7402 cells with 10 µM SLD for 5 min significantly inhibited ERK1/2 phosphorylation under basal conditions (complete medium without FBS) (Fig. 6C). More importantly, another sPLA2 inhibitor, thioetheramide-PC, also significantly reduced basal level of ERK1/2 phosphorylation (data not shown), indicating that sPLA2s are involved in the maintenance of basal ERK1/2 phosphorylation. Third, we tested whether AA could reverse SLD inhibition of basal ERK1/2 phosphorylation. AA pretreatment totally abrogated the inhibitory effect of SLD on ERK1/2 phosphorylation (the percentage of inhibition of ERK1/2 phosphorylation by SLD was 46.4 ± 3.8% in control cells, 1.7 ± 1.2% in 15 min AA-pretreated cells, and 4.5 ± 1.0% in 60-min AA-pretreated cells) (Fig. 6C). These data indicate that SLD inhibits ERK1/2 phosphorylation through sPLA2, and AA is capable of reversing the inhibitory effect of SLD on sPLA2-mediated biological effects, at least ERK1/2 activation. The failure of AA to reverse the inhibitory effect of SLD on Akt implies that SLD inhibition of EGF-stimulated Akt phosphorylation may be independent of sPLA2.
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Overexpression of sPLA2s Did Not Reverse SLD Inhibition of Akt Phosphorylation. To further determine whether the inhibitory effect of SLD on EGFR-mediated Akt phosphorylation was independent of sPLA2, we tested the effect of overexpression of sPLA2s in BEL-7402 cells. Endogenous expression of sPLA2 IB, IIA, V, and X and cPLA2 IV was determined by RT-PCR using specific primers. As shown in Fig. 7A, we detected sPLA2 IIA and cPLA2 IV in BEL-7402 cells. We were not able to detect sPLA2 IB, V, and X even using much more cDNA templates and more PCR cycles (data not shown). These data suggest that sPLA2 IIA may be the main sPLA2 present in BEL-7402 cells. Therefore, we next transiently transfected BEL-7402 cells with sPLA2 IIA or X plasmids. The transfection efficiency was around 15 to 25% as determined by GFP transfection assay. Although the transfection efficiency was not high, the sPLA2 activity as well as mRNA level of sPLA2 IIA and X in transfected cells was markedly increased, and the increased sPLA2 activity in transfected cells was significantly inhibited by SLD (Fig. 7, B and C). In addition, medium collected from sPLA2 IIA or X transfected cells but not from vector transfected cells markedly stimulated the phosphorylation of ERK1/2 (Fig. 7D), demonstrating that sPLA2 IIA and X were produced in transfected cells and secreted into medium. Importantly, overexpression of these sPLA2s reversed SLD inhibition of ERK1/2 phosphorylation (the percentage inhibition of ERK1/2 phosphorylation by SLD in vector, sPLA2 IIA, or X transfected cells was 46.7 ± 6.5%, 14.4 ± 6.0%, and 6.2 ± 3.8%, respectively) (Fig. 7E). These results demonstrated that SLD inhibited ERK1/2 phosphorylation through sPLA2s, which was consistent with the conclusion from AA experiments. However, overexpression of neither sPLA2 IIA nor sPLA2 X reversed SLD (5 µM) inhibition of Akt phosphorylation (Fig. 7F). Of note, the SLD concentration (5 µM) used here was relatively lower compared with the concentrations used in previous experiments, and 5 µM SLD only partially inhibited EGF-stimulated Akt phosphorylation (Fig. 7F). The percentage inhibition of EGF-stimulated Akt phosphorylation by SLD (5 µM) in vector, sPLA2 IIA, or sPLA2 X transfected cells were 60.9 ± 2.0%, 59.9 ± 4.1%, and 60.5 ± 3.7%, respectively) (Fig. 7F). Taken together, these data indicate that SLD inhibition of EGFR-mediated Akt phosphorylation is independent of sPLA2.
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Discussion |
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; Hope et al., 1993; Marshall et al., 1994). In the present study, we also showed that SLD effectively inhibited sPLA2 activity (Fig. 7B). The selective inhibition of sPLA2 by SLD blocked the release of AA, the activation of inducible NO synthase and transcription factor nuclear factor-
B, the production of eicosanoid, and the increase in intracellular Ca2+ in response to stimulators in different cell types (Barnette et al., 1994; Marshall et al., 1994; Thommesen et al., 1998; Baek et al., 2000). Besides these well known effects on phospholipid metabolism, we demonstrated here that SLD dose- and time-dependently inhibited EGF-stimulated Akt phosphorylation in hepatocellular carcinoma BEL-7402 cells as well as in breast cancer MDA-MB-435 cells and human endothelial cells HMEC, and, more importantly, SLD inhibited PDK-1 membrane translocation and PI3K activity. Our results clearly show that inhibition of PI3K/Akt signaling is novel and may be a common pharmacological effect of SLD. This is of importance, given the role of PI3K/Akt signaling in cellular functions. In addition, SLD also inhibited insulin-stimulated Akt phosphorylation, suggesting SLD acts on a target, which is a common event in receptor tyrosine kinase-mediated signaling. However, whether the target is PI3K or the upstream kinases of PI3K remains to be further identified.
Since SLD is an effective inhibitor of sPLA2, and EGF has been shown to stimulate PLA2 activity (Spaargaren et al., 1992; Croxtall et al., 1995; Schalkwijk et al., 1995), it is possible that SLD inhibition of the EGFR/PI3K/Akt signaling pathway is through sPLA2. However, thioetheramide-PC, a structurally modified phospholipid that functions as another specific inhibitor of sPLA2 (Yu et al., 1990; Beck-Speier et al., 2003; Bishop-Bailey and Warner, 2003), did not affect EGFR-mediated Akt phosphorylation, indicating this inhibition is a specific effect of SLD and may be independent of sPLA2. This hypothesis is supported by the following facts. First, SLD inhibition of EGFR-mediated Akt phosphorylation was not mimicked by cyclooxygenase inhibitor indomethacin. Cyclooxygenases are downstream enzymes of PLA2 and convert AA to prostaglandins (Touhey et al., 2002). Second, sPLA2-catalyzed metabolite AA was not able to reverse the inhibitory effect of SLD on EGF-stimulated Akt phosphorylation. Third, overexpression of sPLA2 did not reverse SLD inhibition of Akt phosphorylation. ERK1/2, the other two critical protein kinases in tyrosine kinase receptor-mediated signaling, serve as controls in present study. Although we did not see the inhibitory effect of SLD on EGF-stimulated ERK1/2 phosphorylation, it seems to us that the maintenance of basal activity of ERK1/2 is dependent of sPLA2 since SLD inhibits basal ERK phosphorylation, and this effect is reversed by AA and overexpression of sPLA2s. These findings indicate that both AA and sPLA2 transfection systems work well in our systems and that SLD has both sPLA2-dependent and -independent pharmacological effects in BEL-7402 cells.
Among 10 sPLA2 enzymes that have been described in mammals, group IB, IIA, and V sPLA2 are relatively wide-spread and abundant in tissues (Kudo and Murakami, 2002). We used primers for these three sPLA2 enzymes as well as group X sPLA2 to detect the expression of sPLA2s in BEL-7402 cells by RT-PCR. It was shown that only a 434-bp fragment of sPLA2 IIA was amplified from the total RNA of BEL-7402 cells. Therefore, it is most likely that group IIA but not other sPLA2s exerted effects in BEL-7402 cells. We transfected BEL-7402 cells with group IIA sPLA2, and an around 3-fold increase in sPLA2 activity was observed as determined by sPLA2 activity assay. The assay uses the 1,2-dithio analog of diheptanoyl phosphatidylcholine as a substrate that is specific for most PLA2s (e.g., bee and cobra venoms, pancreatic, etc.) but not for cPLA2. Five micromolar of SLD is a dose that partially, but not completely, inhibited EGF-stimulated Akt phosphorylation. If SLD inhibition of EGFR-mediated Akt activation is dependent on sPLA2, overexpression of group IIA sPLA2 should reverse the inhibition of Akt phosphorylation by 5 µM SLD to some degree. However, neither group IIA nor group X sPLA2 was observed to abrogate the effect of SLD on Akt. Based on the facts mentioned above, we believe that SLD inhibition of EGFR-mediated Akt phosphorylation is independent of sPLA2.
Many PLA2 inhibitors originally thought to be selective for a specific PLA2 are now known to inhibit other isoforms. For example, manoalide has previously been shown to be a potent inhibitor of venom phospholipases A2. Now it reveals that manoalide inhibits both general PLA2 and phosphoinositide-specific phospholipase C (Bennett et al., 1987; Cummings et al., 2000). MAFP, the inhibitor of cPLA2 with an IC50 of 
0.5 µM for purified cPLA2, also inhibits iPLA2 purified from murine macrophage-like P388D1 cells with IC50 of 0.5 µM (Lio et al., 1996). The failure of other PLA2 inhibitors, AACOCF3 and MAFP, and phospholipase C inhibitor U73122
[GenBank]
, to inhibit EGFR-mediated Akt phosphorylation suggests it is not likely that SLD inhibited Akt phosphorylation through inhibiting other isoforms of phospholipases, at least three isoforms, cPLA2, iPLA2, and phospholipase C.
In summary, this study revealed that SLD possessed a novel effect besides its inactivating sPLA2; i.e., SLD inhibits EGFR-mediated Akt activation. This novel effect of SLD is independent of sPLA2. Provided that Akt is a critical mediator in cellular biology, our findings provide important evidence for further exploring the pharmacological effects of SLD.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: EGF, epidermal growth factor; EGFR, EGF receptor; PI3K, phosphatidylinositol 3-kinase; PDK, 3-phosphoinositide-dependent protein kinase; ERK, extracellular signal-regulated kinase; PLA2, phospholipase A2; sPLA2, secretory PLA2; iPLA2, Ca2+-independent PLA2; cPLA2, cytosolic phospholipase A2; SLD, 12-epi-scalaradial; AACOCF3, arachidonyl trifluoromethyl ketone; U73122
[GenBank]
, 1-[6-[[17
-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione; PC, phosphatidylcholine; pEGFP, plasmid vector red-shifted green fluorescence protein; HMEC, human dermal microvascular endothelial cell; FBS, fetal bovine serum; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; RT-PCR, reverse transcriptase-polymerase chain reaction; AA, arachidonic acid; MAFP, methyl arachidomyl fluorophosphonate; GFP, green fluorescence protein.
Address correspondence to: Dr. Liguang Lou, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China. E-mail: lglou{at}mail.shcnc.ac.cn
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