Kenneth J. Serio

Modulation of Pulmonary Leukotriene B4 Production by Cyclooxygenase-2 Inhibitors and Lipopolysaccharide

Purpose: Emerging data continue to link carcinogenesis to inflammatory events involving the eicosanoid metabolic pathways. We therefore evaluated the effects of cyclooxygenase (COX)-2 inhibition on leukotriene (LT) B4 synthesis in the lungs of active smokers, as part of a pilot lung cancer chemoprevention study with celecoxib (Celebrex), an oral COX-2 inhibitor. Experimental Design: Bronchoalveolar lavage was performed before celecoxib treatment and after 1 month of celecoxib treatment to recover alveolar macrophages (AMs) and lining fluid for study. After harvest, AMs were immediately stimulated in vitro with the calcium ionophore A23187. AMs obtained from smokers before treatment and from ex-smoker control subjects were also cultured overnight with SC58236, a selective COX-2 inhibitor, with or without lipopolysaccharide stimulation. Results: Treatment with oral celecoxib only modestly increased LTB4 levels in bronchoalveolar lavage, without increasing the mRNA transcription of 5-lipoxygenase (5-LOX) or 5-LOX-activating protein in AMs, whereas the acute calcium ionophore-stimulated LTB4 production from smokers’ AMs was markedly increased by 10.6-fold. In addition, smokers’ AMs were twice as responsive in producing LTB4 when exposed to lipopolysaccharide compared with ex-smokers’ AMs. Concomitant COX-2 inhibition with SC58236, however, did not significantly impact these changes, whereas the 5-LOX inhibitor Zileuton blocked the generation of LTB4 in a dose-responsive manner. Finally, cycloheximide increased the production of LTB4 under all conditions, suggesting a shunting phenomenon and/or the presence of pathway inhibitors. Conclusions: Our findings suggest that whereas oral celecoxib is capable of modulating LTB4 production in the lung microenvironment, under physiologic conditions, this effect is probably not functionally significant.

White Tea Extract Induces Apoptosis in Non-Small Cell Lung Cancer Cells: the Role of Peroxisome Proliferator-Activated Receptor-γ and 15-Lipoxygenases

Emerging preclinical data suggests that tea possess anticarcinogenic and antimutagenic properties. We therefore hypothesize that white tea extract (WTE) is capable of favorably modulating apoptosis, a mechanism associated with lung tumorigenesis. We examined the effects of physiologically relevant doses of WTE on the induction of apoptosis in non–small cell lung cancer cell lines A549 (adenocarcinoma) and H520 (squamous cell carcinoma) cells. We further characterized the molecular mechanisms responsible for WTE-induced apoptosis, including the induction of peroxisome proliferator-activated receptor-γ (PPAR-γ) and the 15-lipoxygenase (15-LOX) signaling pathways. We found that WTE was effective in inducing apoptosis in both A549 and H520 cells, and inhibition of PPAR-γ with GW9662 partially reversed WTE-induced apoptosis. We further show that WTE increased PPAR-γ activation and mRNA expression, concomitantly increased 15(S)-hydroxy-eicosatetraenoic acid release, and upregulated 15-LOX-1 and 15-LOX-2 mRNA expression by A549 cells. Inhibition of 15-LOX with nordihydroguaiaretic acid (NGDA), as well as caffeic acid, abrogated WTE-induced PPAR-γ activation and upregulation of PPAR-γ mRNA expression in A549 cells. WTE also induced cyclin-dependent kinase inhibitor 1A mRNA expression and activated caspase-3. Inhibition of caspase-3 abrogated WTE-induced apoptosis. Our findings indicate that WTE is capable of inducing apoptosis in non–small cell lung cancer cell lines. The induction of apoptosis seems to be mediated, in part, through the upregulation of the PPAR-γ and 15-LOX signaling pathways, with enhanced activation of caspase-3. Our findings support the future investigation of WTE as an antineoplastic and chemopreventive agent for lung cancer. Cancer Prev Res; 3(9); 1132–40. ©2010 AACR.

Lipopolysaccharide down-regulates the leukotriene C4 synthase gene in the monocyte-like cell line, THP-1

We studied the effects of LPS on cysteinyl leukotriene (LT) synthesis and LTC4 synthase expression in mononuclear phagocytes. Conditioning of the monocyte-like cell line, THP-1, with LPS for 7 days resulted in significantly decreased ionophore-stimulated LTC4 release. The putative LPS receptor, Toll-like receptor 4, was expressed in THP-1 cells. LPS down-regulated LTC4 synthase mRNA in THP-1 cells in a dose- and time-dependent manner, with down-regulation observed as early as 4 h. Conditioning of actinomycin D-treated cells with LPS resulted in no change in the rate of LTC4 synthase mRNA decay. LPS treatment of THP-1 cells, transiently transfected with a LTC4 synthase promoter (1.35 kb)-reporter construct, decreased promoter activity. Neutralization of TNF-α and inhibition of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase did not inhibit the effect of LPS. Treatment of cells with a Toll-like receptor 4-blocking Ab and an inhibitor of NF-κB activation resulted in inhibition of the LPS effect, while activation of NF-κB and p50/p65 overexpression down-regulated the LTC4 synthase gene. LPS down-regulates cysteinyl LT release and LTC4 synthase gene expression in mononuclear phagocytes by an NF-κB-mediated mechanism.

MicroRNA-19a/b mediates grape seed procyanidin extract-induced anti-neoplastic effects against lung cancer ☆

Oncomirs are microRNAs (miRNA) associated with carcinogenesis and malignant transformation. They have emerged as potential molecular targets for anti-cancer therapy. We hypothesize that grape seed procyanidin extract (GSE) exerts antineoplastic effects through modulations of oncomirs and their downstream targets. We found that GSE significantly down-regulated oncomirs miR-19a and -19b in a variety of lung neoplastic cells. GSE also increased mRNA and protein levels of insulin-like growth factor II receptor (IGF-2R) and phosphatase and tensin homolog (PTEN), both predicted targets of miR-19a and -19b. Furthermore, GSE significantly increased PTEN activity and decreased AKT phosphorylation in A549 cells. Transfection of miR-19a and -19b mimics reversed the up-regulations of IGF2R and PTEN gene expression and abrogated the GSE induced anti-proliferative response. Additionally, oral administration of leucoselect phytosome, comprised of standardized grape seed oligomeric procyanidins complexed with soy phospholipids, to athymic nude mice via gavage, significantly down-regulated miR-19a, -19b and the miR-17-92 cluster host gene (MIR17HG) expressions, increased IGF-2R, PTEN, decreased phosphorylated-AKT in A549 xenograft tumors, and markedly inhibited tumor growth. To confirm the absorption of orally administered GSE, plasma procyanidin B1 levels, between 60 and 90 min after gavage of leucoselect phytosome (400 mg/kg), were measured by LC/MS at week 2 and 8 of treatment; the estimated concentration that was associated with 50% growth inhibition (IC50) (1.3 μg/mL) in vitro was much higher than the IC50 (0.032-0.13 μg/ml) observed in vivo. Our findings reveal novel antineoplastic mechanisms by GSE and support the clinical translation of leucoselect phytosome as an anti-neoplastic and chemopreventive agent for lung cancer.

Regulation Of Leukotriene C4 Synthase Gene Expression By Sp1 And Sp3 In Mononuclear Phagocytes

A large body of experimental evidence has implicated the cysteinyl leukotrienes in a variety of inflammatory responses201C;2. Leukotriene C4 (LTC4) synthase (Mr of 16,568; EC#2.5.1.37) is a selective, membrane-bound glutathione-S-transferase that catalyzes the conversion of LTA4 to LTC4. Although LTC4 synthase enzymatic activity is found in various cell types, mRNA for LTC4 synthase is present only in eosinophils, basophils, mast cells, and cells of monocyte/macrophage lineage. Previous studies suggest that LTC4 synthase expression is increased in the bronchial mucosa of patients with aspirin-sensitive asthma3. The gene for LTC4 synthase has been cloned, sequenced, and mapped to the distal region of chromosome 54’5. The LTC4 synthase 5’ flanking region lacks a TATA box but has known consensus sites for ets, AP-1, AP-2, and Sp14’5. Previous work from our laboratory indicates that LTC4 synthase gene expression is upregulated by transforming growth factor-s (TGF-s) through a transcriptional mechanism in the monocyte-like cell line, THP-16. The purpose of this study was to begin to investigate the molecular mechanisms of regulation of transcription of the LTC4 synthase gene in mononuclear phagocytes.