Gallic acid inhibits migration and invasion in human osteosarcoma U-2 OS cells through suppressing the matrix metalloproteinase-2/-9, protein kinase B (PKB) and PKC signaling pathways

Advanced cancer is a multifactorial disease which complicates treatment if the cancer cells have metastasized calling for the targeting of multiple cellular pathways. Gallic acid (GA) is known to possess multiple pharmacological activity including antitumor effects. This study investigated the mechanisms for the anticancer properties of GA on migration and invasion of human osteosarcoma U-2 OS cells. The migration and invasion in U-2 OS cells were determined by a Boyden chamber transwell assay. The expression levels and activities of MMP-2 and MMP-9 were measured by Western blotting, real-time PCR and gelatin zymography assays. All examined proteins levels from Western blotting indicated that GA decreased the protein levels of GRB2, PI3K, AKT/PKB, PKC, p38, ERK1/2, JNK, NF-κB p65 in U-2 OS cells. GA also inhibited the activities of AKT, IKK and PKC by in vitro kinase assay. GA suppressed the migration and invasive ability of U-2 OS cells, and it decreased MMP-2 and MMP-9 protein and mRNA levels and secreted enzyme activities in vitro. These results suggest that potential signaling pathways of GA-inhibited migration and invasion in U-2 OS cells may be due to down-regulation of PKC, inhibition of mitogen-activated protein kinase (MAPK) and PI3K/AKT, resulting in inhibition of MMP-2 and MMP-9 expressions.     

Cardiotoxin III suppresses MDA-MB-231 cell metastasis through the inhibition of EGF/EGFR-mediated signaling pathway

Cardiotoxin III (CTX III), a basic polypeptide isolated from Naja naja atra venom, has been shown to exhibit anticancer activity. Epidermal growth factor (EGF) and its receptor, EGFR, play roles in cancer metastasis in various tumors. We use EGF as a metastatic inducer of MDA-MB-231 cells to investigate the effect of CTX III on cell migration. CTX III inhibited the EGF-induced activation of matrix metalloproteinase-9 (MMP-9), and further suppressed cell invasion and migration without obvious cellular cytotoxicity. CTX III suppressed EGF-induced nuclear factor-kappaB (NF-κB) nuclear translocation and also abrogated the EGF-induced phosphorylation of EGFR, phosphatidylinositol 3-kinase (PI3K)/Akt, and extracellular regulated kinase (ERK)1/2. In addition, CTX III similar to wortmannin (a PI3K inhibitor) and U0126 (an up-stream kinase regulating ERK1/2 inhibitor) attenuated cell migration and invasion induced by EGF. Furthermore, the EGFR inhibitor AG1478 inhibited EGF-induced MMP-9 expression, cell migration and invasion, as well as the activation of ERK1/2 and PI3K/Akt, suggesting that ERK1/2 and PI3K/Akt activation occur downstream of EGFR activation. These findings suggest that CTX III inhibited the EGF-induced invasion and migration of MDA-MB-231 cells via EGFR-dependent PI3K/Akt, ERK1/2, and NF-κB signaling, leading to the down-regulation of MMP-9 expression. These results provide a novel mechanism to explain the role of CTX III as a potent anti-metastatic agent in MDA-MB-231 cells.     

Selaginella tamariscina (Beauv.) possesses antimetastatic effects on human osteosarcoma cells by decreasing MMP-2 and MMP-9 secretions via p38 and Akt signaling pathways

Selaginella tamariscina is a traditional medicinal plant for treatment of some advanced cancers in the Orient. However, the effect of S. tamariscina on metastasis of osteosarcoma and the underlying mechanism remain unclear. We tested the hypothesis that S. tamariscina suppresses cellular motility, invasion and migration and also investigated its signaling pathways. This study demonstrates that S. tamariscina, at a range of concentrations (from 0 to 50 μg/mL), concentration-dependently inhibited the migration/invasion capacities of three osteosarcoma cell lines without cytotoxic effects. Zymographic and western blot analyses revealed that S. tamariscina inhibited the matrix metalloproteinase (MMP)-2 and MMP-9 enzyme activity, as well as protein expression. Western blot analysis also showed that S. tamariscina inhibits phosphorylation of p38 and Akt. Furthermore, SB203580 (p38 inhibitor) and LY294002 (PI3K inhibitor) showed the similar effects as S. tamariscina in U2OS cells. In conclusion, S. tamariscina possesses an antimetastatic activity in osteosarcoma cells by down-regulating MMP-2 and MMP-9 secretions and increasing TIMP-1 and TIMP-2 expressions through p38 and Akt-dependent pathways. S. tamariscina may be a powerful candidate to develop a preventive agent for osteosarcoma metastasis.     

Urocortin induced expression of COX-2 and ICAM-1 via corticotrophin-releasing factor type 2 receptor in rat aortic endothelial cells

Background and purpose:

Our previous study showed that urocortin (Ucn1) exacerbates the hypercoagulable state and vasculitis in a rat model of sodium laurate-induced thromboangiitis obliterans. Furthermore, the inflammatory molecules COX-2 and ICAM-1 may participate in this effect. In the present study, the effects of Ucn1 on COX-2 and ICAM-1 expression in lipopolysaccharide (LPS)-induced rat aortic endothelial cells (RAECs) were investigated and the mechanisms involved explored.

Experimental approach:

RAECs were isolated from adult male Wistar rats, and identified at the first passage. Experiments were performed on cells, from primary culture, at passages 5–8. The expression of COX-2 and ICAM-1 at both mRNA and protein levels was determined by semi-quantitative RT-PCR and Western blot analysis. Levels of PGE₂ and soluble ICAM-1 (sICAM-1) in culture medium were measured by enzyme-linked immunosorbent assay. Furthermore, the phosphorylation status of p38MAPK, ERK1/2, JNK, Akt and NF-κB was analysed by Western blot; nuclear translocation of NF-κB was observed by immunofluorescence.

Key results:

Ucn1 augmented LPS-induced expression of COX-2 and ICAM-1 in RAECs in a time- and concentration-dependent manner. Ucn1 increased PGE₂ and sICAM-1 levels. These effects were abolished by the CRF₂ receptor antagonist, antisauvagine-30, but not by the CRF₁ receptor antagonist, NBI-27914. Moreover, Ucn2 activated p38MAPK and augmented NF-κB nuclear translocation and phosphorylation, whereas ERK1/2, JNK and Akt pathways were not involved in this process.

Conclusions and implications:

These findings suggest that Ucn1 exerts pro-inflammatory effects by augmenting LPS-induced expression of COX-2 and ICAM-1 in RAECs via CRF₂ receptors and the activation of p38MAPK and NF-κB.     

Inhibition of EGF/EGFR activation with naphtho[1,2-b]furan-4,5-dione blocks migration and invasion of MDA-MB-231 cells

Naphtho[1,2-b]furan-4,5-dione (NFD), a bioactive component of Avicennia marina, has been demonstrated to display anti-cancer activity. Activation of epidermal growth factor receptor (EGFR)-induced signaling pathway has been correlated with cancer metastasis in various tumors, including breast carcinoma. We use EGF as a metastatic inducer of MDA-MB-231 cells to investigate the effect of NFD on cell migration and invasion. NFD suppressed EGF-mediated protein levels of c-Jun and c-Fos, and reduced MMP-9 expression and activity, concomitantly with a marked inhibition on cell migration and invasion without obvious cellular cytotoxicity. NFD abrogated EGF-induced phosphorylation of EGF receptor (EGFR) and phosphatidylinositol 3-kinase (PI3K)/Akt. The specific PI3K inhibitor, wortmannin, blocked significantly EGF-induced cell migration and invasion. Furthermore, the EGFR inhibitor AG1478 inhibited EGF-induced MMP-9 expression, cell migration and invasion, as well as the activation of PI3K/Akt, suggesting that PI3K/Akt activation occur downstream of EGFR activation. These findings suggest that NFD inhibited the EGF-induced invasion and migration of MDA-MB-231 cells via EGFR-dependent PI3K/Akt signaling, leading to the down-regulation of MMP-9 expression. These results provide a novel mechanism to explain the role of NFD as a potent anti-metastatic agent in MDA-MB-231 cells.     

Leptin protection of salivary gland acinar cells against ethanol cytotoxicity involves Src kinase-mediated parallel activation of prostaglandin and constitutive nitric oxide synthase pathways

Leptin, a pleiotropic cytokine secreted by adipocytes but also identified in salivary glands and saliva, is recognized as an important element of oral mucosal defense. Here, we report that in sublingual salivary glands leptin protects the acinar cells of against ethanol cytotoxicity. We show that ethanol- induced cytotoxicity, characterized by a marked drop in the acinar cell capacity for NO production, arachidonic acid release and prostaglandin generation, was subject to suppression by leptin. The loss in countering capacity of leptin on the ethanol-induced cytotoxicity was attained with cyclooxygenase inhibitor, indomethacin and nitric oxide synthase (cNOS) inhibitor, L-NAME, as well as PP2, an inhibitor of Src kinase. Indomethacin, while not affecting leptin-induced arachidonic acid release, caused the inhibition in PGE₂ generation, pretreatment with L-NAME led to the inhibition in NO production, whereas PP2 exerted the inhibitory effect on leptin-induced changes in NO, arachidonic acid, and PGE₂. The leptin-induced changes in arachidonic acid release and PGE₂ generation were blocked by ERK inhibitor, PD98059, but not by PI3K inhibitor, wortmannin. Further, leptin suppression of ethanol cytotoxicity was reflected in the increased Akt and cNOS phosphorylation that was sensitive to PP2. Moreover, the stimulatory effect of leptin on the acinar cell cNOS activity was inhibited not only by PP2, but also by Akt inhibitor, SH-5, while wortmannin had no effect. Our findings demonstrate that leptin protection of salivary gland acinar cells against ethanol cytotoxicity involves Src kinase-mediated parallel activation of MAPK/ERK and Akt that result in up-regulation of the respective prostaglandin and nitric oxide synthase pathways. Received 1 August 2007; accepted 10 October 2007     

Modulation of metalloproteinase-9 in U87MG glioblastoma cells by A3 adenosine receptors

In this work, we investigated the biological functions of adenosine (ado) in metalloproteinase-9 (MMP-9) regulation in U87MG human glioblastoma cells. The nucleoside was able to increase both MMP-9 mRNA and protein levels through A₃ receptors activation. We revealed that A₃ receptor stimulation induced an increase of MMP-9 protein levels in cellular extracts of U87MG cells by phosphorylation of extracellular signal-regulated protein kinases (ERK1/2), c-Jun N-terminal kinase/stress-activated protein kinase (pJNK/SAPK), protein kinase B (Akt/PKB) and finally activator protein 1 (AP-1). A₃ receptor activation stimulated also an increase of extracellular MMP-9 in the supernatants from U87MG glioblastoma cells. Finally, the Matrigel invasion assay demonstrated that A₃ receptors, by inducing an increase in MMP-9 levels, was responsible for an increase of glioblastoma cells invasion. Collectively, these results suggest that ado, through A₃ receptors activation, modulates MMP-9 protein levels and plays a role in increasing invasion of U87MG cells.     

Camptothecin suppresses expression of matrix metalloproteinase-9 and vascular endothelial growth factor in DU145 cells through PI3K/Akt-mediated inhibition of NF-κB activity and Nrf2-dependent induction of HO-1 expression

Though camptothecin (CPT) possesses potent anti-inflammatory, immunomodulatory, anticancerous, and antiproliferative effects, little is known about the mechanism by which CPT regulates the expression of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF). Therefore, the current study aimed to investigate the effects of CPT on the expression of MMP-9 and VEGF, which are important factors for the invasion of tumors. In vitro application of CPT resulted in a slight inhibition of cell proliferation and a significant reduction in the matrigel invasion of DU145 cells. Treatment with CPT also downregulated phorbol-12-myristate-13-acetate (PMA)- and tumor necrosis factor-α (TNF-α)-induced MMP-9 and VEGF expression by inhibiting nuclear factor-κB (NF-κB) activity. Downregulation of phosphoinositide 3-kinase (PI3K)/Akt phosphorylation in response to CPT was revealed as an upstream pathway regulating the expression of MMP-9 and VEGF accompanying the inhibition of NF-κB activity. We further confirmed that CPT inhibits PMA-induced MMP-9 and VEGF expression by upregulating nuclear factor-erythroid related factor-2 (Nrf2)-mediated heme oxygenase-1 (HO-1) induction. Taken together, these data indicate that CPT inhibits the invasion of cancer cells accompanied by suppression of MMP-9 and VEGF production by suppressing the PI3K/Akt-mediated NF-κB pathway and enhancing the Nrf2-dependent HO-1 pathway, suggesting that CPT may be a good candidate to inhibit MMP-9 and VEGF expression.     

The extraneuronal transport mechanism for noradrenaline (uptake2) avidly transports 1-methyl-4-phenylpyridinium (MPP+)

Summary The corticosterone-sensitive extraneuronal transport mechanism for noradrenaline (uptake₂) removes the neurotransmitter from the extracellular space. Recently, an experimental model for uptake₂ has been introduced which is based on tissue culture techniques (human Caki-1 cells). The present study describes some properties of uptake₂ in Caki-1 cells and introduces a new substrate, i.e., 1-methyl-4-phenylpyridinium (MPP⁺).Experiments on Caki-1 cells disclosed disadvantages of tritiated noradrenaline as substrate for the investigation of uptake₂. The initial rate of ³H-noradrenaline transport [k_in = 0.58 l/(mg protein · min)] was low compared with other cellular transport systems and intracellular noradrenaline was subject to rapid metabolism (k_{O-methylation} = 0.54 min^–1). The neurotoxin MPP⁺ was found to be a good substrate of uptake₂. Initial rates of specific ³H-MPP⁺ transport into Caki-1 cells were saturable, the K_m being 24 mol/l and the V_max being 420 pmol/(mg protein · min). The rate constant of specific inward transport was 34 times higher [19.6 mol/l (mg protein · min)] than that of ³H-noradrenaline. The ratio specific over non-specific transport was considerably higher for ³H-MPP⁺ (12.6) than for ³H-noradrenaline (3.0). ³H-MPP⁺ transport into Caki-1 cells was inhibited by various inhibitors of uptake₂. The highly significant positive correlation (p < 0.001, r = 0.986, n = 7) between the IC₅₀'s for the inhibition of the transport of ³H-noradrenaline and ³H-MPP⁺, respectively, proves the hypothesis that MPP⁺ enters Caki-1 cells via uptake₂. ³H-MPP⁺ is taken up via uptake₂ not only by Caki-1 cells but also by the isolated perfused rat heart which is another established model of uptake₂.Tritiated MPP⁺ is a new and convenient tool for the investigation of uptake₂. The rate constant for inward transport, the factor of accumulation and the ratio specific over non-specific transport are considerably higher for ³H-MPP⁺ than for ³H-noradrenaline. In uptake studies with ³H-MPP⁺ inhibition of intracellular noradrenaline-metabolizing enzymes is not necessary. In tissues and tissue cultures which possess fewer uptake₂ carriers than Caki-1 cells or the rat heart, the identification and characterization of uptake₂ can be expected to be greatly facilitated by the use of ³H-MPP⁺.Supported by the Deutsche Forschungsgemeinschaft (SFB 176) Send offprint requests to H. Russ at the above address     

o,p'-DDT induces cyclooxygenase-2 gene expression in murine macrophages: Role of AP-1 and CRE promoter elements and PI3-kinase/Akt/MAPK signaling pathways

Dichlorodiphenyltrichloroethane (DDT) has been used as an insecticide to prevent the devastation of malaria in tropical zones. However, many reports suggest that DDT may act as an endocrine disruptor and may have possible carcinogenic effects. Cyclooxygenase-2 (COX-2) acts as a link between inflammation and carcinogenesis through its involvement in tumor promotion. In the present study, we examined the effect of o,p′-DDT on COX-2 gene expression and analyzed the molecular mechanism of its activity in murine RAW 264.7 macrophages. Exposure to o,p′-DDT markedly enhanced the production of prostaglandin E₂ (PGE₂), a major COX-2 metabolite, in murine macrophages. Furthermore, o,p′-DDT dose-dependently increased the levels of COX-2 protein and mRNA. Transfection with human COX-2 promoter construct, electrophoretic mobility shift assays and DNA-affinity protein-binding assay experiments revealed that o,p′-DDT activated the activator protein 1 (AP-1) and cyclic AMP response element (CRE) sites, but not the NF-κB site. Phosphatidylinositol 3 (PI3)-kinase, its downstream signaling molecule, Akt, and mitogen-activated protein kinases (MAPK) were also significantly activated by the o,p′-DDT-induced AP-1 and CRE activation. These results demonstrate that o,p′-DDT induced COX-2 expression via AP-1 and CRE activation through the PI3-K/Akt/ERK, JNK, and p38 MAP kinase pathways. These findings provide further insight into the signal transduction pathways involved in the carcinogenic effects of o,p′-DDT.     

Prostanoid EP1 receptor as the target of (?)-epigallocatechin-3-gallate in suppressing hepatocellular carcinoma cells in vitro

Aim:

To investigate the effects of (−)-epigallocatechin-3-gallate (EGCG), an active compound in green tea, on prostaglandin E₂ (PGE₂)-induced proliferation and migration, and the expression of prostanoid EP₁ receptors in hepatocellular carcinoma (HCC) cells.

Methods:

HCC cell line HepG2, human hepatoma cell lines MHCC-97L, MHCC-97H and human hepatocyte cell line L02 were used. Cell viability was analyzed using MTT assay. PGE₂ production was determined with immunoassay. Wound healing assay and transwell filter assay were employed to assess the extent of HCC cell migration. The expression of EP₁ receptor and Gq protein were examined using Western blot assay.

Results:

PGE₂ (4-40000 nmol/L) or the EP₁ receptor agonist ONO-DI-004 (400-4000 nmol/L) increased the viability and migration of HepG2 cells in concentration-dependent manners. EGCG (100 μg/mL) significantly inhibited the viability and migration of HepG2 cells induced by PGE₂ or ONO-DI-004. HepG2 cells secreted an abundant amount of PGE₂ into the medium, and EGCG (100 μg/mL) significantly inhibited the PGE₂production and EP₁ receptor expression in HepG2 cells. EGCG (100 μg/mL) also inhibited the viability of MHCC-97L cells, but not that of MHCC-97H cells. Both EGCG (100 μg/mL) and EP₁ receptor antagonist ONO-8711 inhibited PGE₂ 4 μmol/L and ONO-DI-004 400 nmol/L-induced growth and migration of HepG2 cells. Both EGCG (100 μg/mL) and ONO-8711 210 nmol/L inhibited PGE₂- and ONO-DI-004-induced EP₁ expression. EGCG and ONO-8711 had synergistic effects in inhibiting EP₁ receptor expression. PGE₂, ONO-DI-004, ONO-8711, and EGCG had no effects on Gq expression in HepG2 cells, respectively.

Conclusion:

These findings suggest that the anti-HCC effects of EGCG might be mediated, at least partially, through the suppressing EP₁ receptor expression and PGE₂ production.     

小檗碱通过调控PI3K/Akt通路抑制喉癌细胞生长和转移

目的探讨小檗碱对喉癌Hep2细胞增殖、凋亡和侵袭的影响以及相关作用机制。方法培养Hep2细胞分为空白对照组和5、10、20μmol·L-1小檗碱组,CCK-8检测Hep2的增殖活性,流式细胞术检测细胞凋亡情况,划痕实验检测细胞迁移情况,Transwell法检测细胞侵袭能力,Western blot法检测凋亡蛋白酶激活因子1(Apaf1)、剪切后半胱天冬酶(cl-caspase)-9、cl-caspase-3蛋白、血管内皮生长因子(VEGF)、基质金属蛋白酶(MMP)-2、MMP-9、磷脂酰肌醇3激酶(PI3K)、磷酸化蛋白激酶B(p-Akt)和p-p65蛋白表达水平。结果与空白对照组相比,5、10、20μmol·L-1小檗碱组细胞增殖活性显著降低(P <0.01);凋亡细胞百分比显著增加(P <0.01),Apaf1、cl-caspase-9和cl-caspase-3蛋白水平显著上调(P <0.01);划痕愈合率显著降低(P <0.01),侵袭细胞数目显著减少(P <0.01);细胞中VEGF、MMP-2、MMP-9、PI3K、p-Akt、p-p65蛋白水平显著降低(P <0.01),且均呈浓度依赖性。结论小檗碱通过调控PI3K/Akt通路抑制喉癌细胞增殖、侵袭和迁移,诱导细胞凋亡。     

AKT signaling is involved in fucoidan-induced inhibition of growth and migration of human bladder cancer cells

We identified a novel mechanism of AKT signaling in the fucoidan-induced proliferation and migration of human urinary 5637 cancer cells. Fucoidan treatment showed a significant growth inhibition followed by G₁-phase-associated up-regulation of p21WAF1 expression and suppression of cyclins and CDK expression in 5637 cells. Also, fucoidan treatment induced the activation of AKT signaling, which was inhibited by treatment with wortmannin, a PI3K-specific inhibitor. Blockade of the AKT function reversed the fucoidan-mediated inhibition of cell proliferation, the increased G₁-phase-associated p21WAF1 expression, and the reduction of cell-cycle proteins. Moreover, treatment with fucoidan blocked migration and invasion of 5637 cells. This inhibition was attributed to decreased expression of MMP-9, which was mediated by down-regulation of AP-1 and NF-κB binding activity. Furthermore, wortmannin treatment abolished the decreased cell migration and invasion and the inhibition of MMP-9 expression via the suppression of NF-κB and AP-1 in fucoidan-treated cells. Similar results were observed in another bladder cancer T-24 cells treated with fucoidan. Finally, overexpression of the AKT gene inhibited the proliferation, migration and invasion of bladder cancer cells. These data suggest that the activation of AKT signaling is involved in growth inhibition and suppression of the migration and invasion of bladder cancer cells treated with fucoidan.     

Cholesterol-3-beta, 5-alpha, 6-beta-triol induced PI3K-Akt-eNOS-dependent cyclooxygenase-2 expression in endothelial cells

Oxidized cholesterols belong to a subgroup of oxLDLs which play major roles in atherosclerosis. In order to investigate the contribution of oxysterols from oxLDLs in atherosclerosis, cholesterol-3-beta, 5-alpha, 6-beta-triol (α-Triol) was studied in human umbilical vein endothelial cells. We found that α-Triol concentration- and time-dependently enhanced COX-2 protein expression and mRNA production followed by PGE₂ generation in human umbilical vein endothelial cells. In addition, α-Triol upregulated peNOS¹¹⁷⁷ protein phosphorylation and concentration-dependently increased nitric oxide production. eNOS¹¹⁷⁷ phosphorylation was abrogated by the PI3K inhibitor, LY294002. In studying the mechanisms involved in α-Triol-induced COX-2/PGE₂ production, inhibitors of NOS, PI3K, p38, and NF-κB, effectively attenuated COX-2 protein induction and mRNA expression, suggesting that the PI₃K-Akt-eNOS pathway, p38MAPK, and NF-κB are involved in α-Triol-induced COX-2 expression, and following increases in p38 and Akt phosphorylation, the concentration-dependent inhibition of COX-2 protein expression by L-NAME further suggested their involvement at the translation level. We concluded that α-Triol increases COX-2 mRNA and protein expression via coordination with the PI₃K-Akt-eNOS pathway and NF-κB. Moreover, COX-2 gene expression might be regulated by activated p38 MAPK in another unknown regulation pathway. Our findings also suggested that α-Triol might contribute to the effect of induced atherosclerosis in humans through COX-2 production in endothelial cells.     

The role of cyclooxygenase-2-dependent signaling via cyclic AMP response element activation on aromatase up-regulation by o,p′-DDT in human breast cancer cells

o,p′-Dichlorodiphenyltrichloroethane (o,p′-DDT) is a DDT isomer and xenoestrogen that can induce inflammation and cancer. However, the effect of o,p′-DDT on aromatase is unclear. Thus, we investigated the effects of o,p′-DDT on aromatase expression in human breast cancer cells. We also examined whether cyclooxygenase-2 (COX-2) is involved in o,p′-DDT-mediated aromatase expression. Treatment with o,p′-DDT-induced aromatase protein expression in MCF-7 and MDA-MB-231 human breast cancer cells; enhancing aromatase gene expression, and enzyme and promoter activity. Treatment with ICI 182.780, a estrogen receptor antagonist, did not affect the inductive effects of o,p′-DDT on aromatase expression. In addition, o,p′-DDT increased COX-2 protein levels markedly, increased COX-2 mRNA expression and promoter activity, enhanced the production of prostaglandin E₂ (PGE₂), induced cyclic AMP response element (CRE) activation, and cAMP levels and binding of CREB. o,p′-DDT also increased the phosphorylation of PKA, Akt, ERK, and JNK in their signaling pathways in MCF-7 and MDA-MB-231 cells. Finally, o,p′-DDT induction of aromatase was inhibited by various inhibitors [COX-2 (by NS-398), PKA (H-89), PI3-K/Akt (LY 294002), EP2 (AH6809), and EP4 receptor (AH23848)]. Together, these results suggest that o,p′-DDT increases aromatase, and that o,p′-DDT-induced aromatase is correlated with COX-2 up-regulation, mediated via the CRE activation and PKA and PI3-kinase/Akt signaling pathways in breast cancer cells.     

The effect of inhibition of Ca2+-independent phospholipase A2 on chemotherapeutic-induced death and phospholipid profiles in renal cells

We demonstrate that cells derived from primary cultures of rabbit proximal tubules (RPTC), human embryonic kidney (HEK293) and human kidney carcinomas (Caki-1) express microsomal Ca(2+)-independent phospholipase A(2) (iPLA(2)gamma) and cytosolic Ca(2+)-independent phospholipase A(2) (iPLA(2)beta). Inhibition of iPLA(2) activity in these cells using the iPLA(2) inhibitor bromoenol lactone (BEL) (0-5.0microM) for 24h did not induce cell death as determined by annexin V and propidium iodide (PI) staining. However, BEL treatment prior to cisplatin (50muM) or vincristine (2microM) exposure reduced apoptosis 30-50% in all cells tested (RPTC, HEK293 and Caki-1 cells). To identify the phospholipids altered during cell death electrospray ionization-mass spectrometry and lipidomic analysis of HEK293 and Caki-1 cells was performed. Cisplatin treatment reduced 14:0-16:0 and 16:0-16:0 phosphatidylcholine (PtdCho) 50% and 35%, respectively, in both cell lines, 16:0-18:2 PtdCho in Caki-1 cells and increased 16:1-22:6 plasmenylcholine (PlsCho). BEL treatment prior to cisplatin exposure further decreased 14:0-16:0 PtdCho, 16:0-16:1 PlsCho and 16:0-18:1 PlsCho in HEK293 cells, and inhibited cisplatin-induced increases in 16:1-22:6 PlsCho in Caki-1 cells. Treatment of cells with BEL prior to cisplatin exposure also increased the levels of several arachidonic containing phospholipids including 16:0-20:4, 18:1-20:4, and 18:0-20:4 PtdCho, compared to cisplatin only treated cells. These data demonstrate that inhibition of iPLA(2) protects against chemotherapeutic-induced cell death in multiple human renal cell models, identifies specific phospholipids whose levels are altered during cell death, and demonstrates that alterations in these phospholipids correlate to the protection against cell death in the presence of iPLA(2) inhibitors.