Suppression Of IL-8 Research Articles

Low G9a expression is a tumor progression factor of colorectal cancer via IL-8 promotion.

The histone methyltransferase G9a is expressed in various types of cancer cells, including colorectal cancer (CRC) cells. Interleukin 8 (IL)-8, also known as C-X-C motif chemokine ligand 8 (CXCL8), is a chemokine that plays a pleiotropic function in the regulation of inflammatory responses and cancer development. Here, we examined the relationship between G9a and IL-8 and the clinical relevance of this association. We immunohistochemically analyzed 235 resected CRC samples to correlate clinical features. Samples with high G9a expression had better overall survival and relapse-free survival than those with low G9a expression. Univariate and multivariate analyses demonstrated that low G9a expression remained a significant independent prognostic factor for increased disease recurrence and decreased survival (P < 0.05). G9a was expressed at high levels in commercially available CRC cell lines HCT116 and HT29. Knockdown of G9a by siRNA, shRNA or the G9a-specific inhibitor BIX01294 upregulated IL-8 expression. The number of spheroids was significantly increased in HCT116 cells with stably suppressed G9a expression, and the number of spheroids was significantly decreased in HCT116 cells with stably suppressed IL-8 expression. Thus, the suppression of IL-8 by G9a may result in a better prognosis in CRC cases with high G9a expression. Furthermore, G9a may suppress cancer stemness and increase chemosensitivity by controlling IL-8. Therefore, G9a is a potential novel marker for predicting CRC prognosis, and therapeutic targeting of G9a in CRC should be controversial.

Impact of First-Line Antimicrobials on Chlamydia trachomatis-Induced Changes in Host Metabolism and Cytokine Production.

Urogenital infections with Chlamydia trachomatis (C. trachomatis) are the most common bacterial sexually transmitted diseases worldwide. As an obligate intracellular bacterium, chlamydial replication and pathogenesis depends on the host metabolic activity. First-line antimicrobials such as doxycycline (DOX) and azithromycin (AZM) have been recommended for the treatment of C. trachomatis infection. However, accumulating evidence suggests that treatment with AZM causes higher rates of treatment failure than DOX. Here, we show that an inferior efficacy of AZM compared to DOX is associated with the metabolic status of host cells. Chlamydial metabolism and infectious progeny of C. trachomatis were suppressed by therapeutic relevant serum concentrations of DOX or AZM. However, treatment with AZM could not suppress host cell metabolic pathways, such as glycolysis and mitochondrial oxidative phosphorylation, which are manipulated by C. trachomatis. The host cell metabolic activity was associated with a significant reactivation of C. trachomatis after removal of AZM treatment, but not after DOX treatment. Furthermore, AZM insufficiently attenuated interleukin (IL)-8 expression upon C. trachomatis infection and higher concentrations of AZM above therapeutic serum concentration were required for effective suppression of IL-8. Our data highlight that AZM is not as efficient as DOX to revert host metabolism in C. trachomatis infection. Furthermore, insufficient treatment with AZM failed to inhibit chlamydial reactivation as well as C. trachomatis induced cytokine responses. Its functional relevance and the impact on disease progression have to be further elucidated in vivo.

SUN-LB27 Interleukin-8 - a Possible Target for Melanoma Treatment? In-Vitro Studies Based on Human Melanoma Cell Models

Previous clinical studies showed that menstruating females were better protected in melanoma than post-menopausal women and men of any age. In addition, epidemiological studies showed an increased male mortality in melanoma. But these studies did not correlate with steroid status in females. Our in-vitro study showed female sex hormone progesterone significantly inhibited human melanoma cell growth. Further in-vitro study showed that progesterone action was mediated by a specific suppression of pro-inflammatory cytokine IL-8. Our research also showed that addition of IL-8 (1 ng/ml) to melanoma cells stimulated cell growth (117%) and suppression of IL-8 by curcumin (100 μM) pre-treatment suppressed human melanoma cell growth (26%) in-vitro. This observation prompted us to check the effect of male sex hormones androstenedione (AD) and testosterone (T) on melanoma cell growth. AD and T also suppressed cell growth and IL-8 secretion, but not as significantly as that of progesterone. However, addition of progesterone (10 μM) along with androgens showed an additive effect on the inhibition of melanoma cell growth and suppression of IL-8 secretion. As steroids (P, AD, T) targeted IL-8 for their action, it was decided to check whether vitamin-D3 also targeted IL-8 secretion and cell growth. Active form of vit-D3 (25 μM) also suppressed IL-8 secretion and cell growth. But, addition of progesterone (50 μM) along with D3 significantly suppressed cell growth and IL-8 secretion. This brought IL-8 into focus as a key molecule regulating melanoma cell growth. In order to check whether IL-8 was the molecule involved in regulating melanoma cell growth, IL-8 rescue experiment after curcumin (25 μM) pre-treatment was carried out. IL-8 (100 ng/ml) was able to rescue cell growth completely after pre-treatment with curcumin, suggesting IL-8 was the molecule involved in regulating melanoma cell growth. Literature also suggested important role for IL-8 in regulating melanoma cell growth. Conditional expression of IL-8 in nude mouse by Dr. Singh et al., indicated in-vivo role of IL-8 in melanoma growth and metastasis. Conclusion: Both, in-vitro and in-vivo studies suggested an important role for IL-8 in regulating melanoma growth and metastasis. So, IL-8 could be targeted to arrest melanoma growth and metastasis in-vivo. Hence, IL-8 could be a potential target for melanoma treatment.

Can Interleukin‐8 Alone be Responsible for Regulating Melanoma Growth &amp; Metastasis? In‐vitro studies based on treatments of human melanoma cell lines (BLM and 1205Lu) with sex steroids, IL‐8, curcumin and active vitamin‐D3

Previous clinical studies indicated that menstruating females were better protected in melanoma than post‐menopausal women and men of any age. But, the mechanism responsible for protection in menstruating females was not investigated. In addition, epidemiological data showed an increased male mortality in melanoma. Our in‐vitro study showed female sex hormone progesterone significantly inhibited human melanoma cell growth. Elisarray showed that progesterone action was mediated by a specific suppression of pro‐inflammatory cytokine IL‐8. So, the effect of male sex hormones androstenedione (AD) and testosterone (T) on melanoma cell growth was checked. AD and T also suppressed cell growth and IL‐8 secretion. But, addition of progesterone (10μM) along with androgens showed an additive effect on the inhibition of melanoma cell growth and IL‐8 suppression. This observation gave the idea of protective function of progesterone in melanoma, which was experimentally observed by a significant decrease in in‐vitro migration and adhesion functions. As steroids (P, AD, T) targeted IL‐8 for their actions, effect of vit‐D3 on melanoma cells was checked. Active form of vitamin‐D3 (25 μM) also targeted IL‐8 and decreased cell growth, suggesting IL‐8 could be regulating melanoma cell growth. In order to check whether IL‐8 regulated melanoma cell growth, IL‐8 was directly added to melanoma cells and endogenous IL‐8 was suppressed by 2 hrs pre‐treatment with curcumin. Addition of IL‐8 (1 ng/ml) to 1205Lu melanoma cells stimulated cell growth to 117%, whereas suppression of IL‐8 by curcumin (100 μM) pre‐treatment decreased cell growth to 24%. Similar results were obtained with BLM cells. This brought IL‐8 into focus as an important molecule regulating melanoma cell growth. Further in‐vitro experiments to restore curcumin (25 μM) pre‐treated melanoma cell growth (71.5%) by the addition of IL‐8 (100 ng/ml) resulted in a near complete restoration of cell growth (95.3%) in 1205Lu melanoma cells, suggesting IL‐8 could be the molecule regulating melanoma cell growth. Similar results were observed with BLM cells. In addition, in‐vivo conditional expression of IL‐8 in nude mouse by Dr. Singh et al., indicated involvement of IL‐8 in melanoma growth and metastasis.ConclusionBoth in‐vitro and in‐vivo studies indicated that IL‐8 alone could be responsible for regulating melanoma growth and metastasis. Hence, IL‐8 could be targeted to arrest melanoma growth and metastasis in‐vivo.

Myricetin from Rhodomyrtus tomentosa (Aiton) Hassk fruits attenuates inflammatory responses in histamine-exposed endothelial cells

Histamine plays a key role in inflammatory responses via increasing chemokine and adhesion molecule productions and augmenting vascular permeability in endothelial cells. Rhodomyrtus tomentosa has been found as a rich source of structurally diverse and biologically active metabolites. In this study, the role of phenolic compound from Rhodomyrtus tomentosa (Aiton) Hassk fruits in down-regulation of histamine-induced EA.hy926 endothelial cell activation was investigated. Herein, myricetin was successfully isolated from R. tomentosa fruits by HPLC and its characterization was identified by mass spectrometer and nuclear magnetic resonance spectroscopy. It was revealed that myricetin was effective in suppression of IL-8 and MCP-1 productions and adhesion molecule generation. Moreover, NF-κB activation was inhibited by myricetin via reducing IκB-α phosphorylation and p50/p65 subunit level. Notably, myricetin potentially attenuated vascular permeability of endothelial cells through decrease in eNOS phosphorylation and intracellular calcium elevation. These results indicate that myricetin from R. tomentosa possesses a promissing pharmaceutical property for the amelioration of endothelial inflammatory responses.

Inula helenium and Grindelia squarrosa as a source of compounds with anti-inflammatory activity in human neutrophils and cultured human respiratory epithelium.

During the epidemic season, over 90% of acute wheezing disease is associated with bronchial inflammation. Both neutrophil- and eosinophil-mediated inflammation have been involved in the pathophysiology of acute bronchitis, but neutrophil cell recruitment has been shown to be dominant. The ongoing inflammation increases the chemotaxis of neutrophils to inflamed site providing to their overaccumulation. The pharmacological reduction of neutrophil migration can be limited by suppression of major chemo-attractants and cytokines (IL-8, IL-1β and TNF-α) release and downregulation of adhesive molecules. During a screening of plants traditionally used in respiratory tracts diseases (e.g. cough, rhinitis, bronchitis, throat infection, fever, influenza) in Europe, we have selected roots of Inula helenium and aerial parts of Grindelia squarrosa as a potential source of compounds limiting neutrophil migration. The effect on IL-8, IL-1β and TNF-α release by neutrophils and respiratory epithelium cell line (A549) was measured by enzyme-linked immunosorbent assay (ELISA). The surface expression of adhesive molecules was analyzed with flow cytometry, and the neutrophil attachment to the epithelial cells was assessed fluorimetrically. We confirmed the ability of selected extracts and compounds to suppress neutrophil binding to the epithelium surface via downregulation of β2 integrin. Alantolactone and grindelic acid have shown significant suppression of IL-8, TNF-α and IL-1β release comparable with budesonide, used as a positive control. The present study demonstrated that Inula helenium and Grindelia squarrosa, which have been traditionally used in Europe as medicinal plants, are a valuable source of active compounds with anti-inflammatory activity. Our observations justify the traditional use of I.helenium and G.squarrosa for a treatment of inflammation-based diseases in respiratory tract.

MON-312 Biochemical Basis of Steroid Hormones Action on Human Melanoma Cell Growth In-Vitro and Its Clinical Implication

: Clinical study showed that menstruating females were better protected in melanoma than post-menopausal women and men of any age. However, the study did not correlate with steroid status in females. Literature showed that progesterone (P) level varied in menstruating females between 1000 to 1500 ng/dL. Whereas in post-menopausal women, progesterone ranged between 20 to 100 ng/dL and in males between 27 - 90 ng/dL. Our earlier in-vitro research showed that progesterone significantly inhibited human melanoma (BLM) cell growth. So, it was hypothesized that progesterone could be the molecule protecting menstruating females in melanoma. Further research showed that androgens (androstenedione, AD and testosterone, T) also inhibited human melanoma cell growth in-vitro, but only at high concentrations (100 μM and 200 μM). Co-incubation of AD and T separately with P showed an additive effect on human melanoma cell growth inhibition with as low as 10 μM concentration of progesterone, suggesting the protective function of progesterone. As literature showed that progesterone action was mediated through cytokines, Elisarray was used to check the biochemical basis of P, AD and T actions on BLM cells. Elisarray showed that there was a specific suppression of IL-8 cytokine in the supernatants of AD, P and AD+P co-incubated cells compared to control cells. Decrease in IL-8 was quantitated by Elisa, which displayed a direct correlation with decrease in BLM cell growth. In order to confirm the above findings, all the experiments were repeated on another human melanoma 1205Lu cell line. There was a specific suppression of IL-8 in the supernatants, as shown by both Elisarray and IL-8 Elisa. To further consolidate the findings, action of another androgen T, P and T+P were checked on both BLM and 1205Lu cell lines. Again a specific suppression of IL-8 was observed in the supernatants by Elisarray. In addition, Kanda and Watanabe already showed that dihydrotestosterone and estrogen also suppressed IL-8 in melanoma cells. Conclusion: All the tested steroids specifically suppressed IL-8, resulting in the suppression of melanoma cell growth. So, IL-8 could be playing an important role in melanoma growth. In fact, our in-vitro study involving curcumin pre-treatment already showed that IL-8 was the molecule involved in the regulation of human melanoma cell growth. So, IL-8 could be considered as a target for melanoma treatment.

Profiling the immunotoxicity of chemicals based on in vitro evaluation by a combination of the Multi-ImmunoTox assay and the IL-8 Luc assay.

We established a luciferase reporter assay system, the Multi-ImmunoTox Assay (MITA), which can evaluate the effects of chemicals on the promoter activities of four cytokines: IL-2, IFN-γ, IL-1β, and IL-8. We previously reported that MITA correctly reflected the change in mRNA of human whole-blood cells treated with dexamethasone, cyclosporine, FK506, or several other immunosuppressive drugs. In this study, we combined MITA with the IL-8 Luc assay to detect skin sensitization chemicals (OECD 442E) (modified MITA: mMITA) and established a data set of 60 chemicals examined by mMITA. Using the mMITA results, chemicals can be classified based on the lowest observed effect level (LOEL) of chemicals in suppressing or augmenting the promoter activities of the four cytokines. Moreover, we demonstrated that K-means clustering and hierarchical clustering of the 60 chemicals based on the LOEL for their effects on IL-2 and IL-8 promoter activities and the judgment by the IL-8 Luc assay resulted in the same 6-cluster solution: cluster 1 with preferential suppression of IL-8, cluster 2 with suppression of IL-2 and a positive IL-8 Luc assay result, cluster 3 with suppression of both IL-2 and IL-8, cluster 4 with no effects on IL-2 or IL-8 and a negative IL-8 Luc assay result, cluster 5 with suppression of both IL-2 and IL-8 and a negative IL-8 Luc assay result, and cluster 6 with preferential suppression of IL-8. These data suggest that mMITA is a promising novel high-throughput approach for detecting unrecognized immunological effects of chemicals and for profiling their immunotoxic effects.

Compound A attenuates toll-like receptor 4-mediated paclitaxel resistance in breast cancer and melanoma through suppression of IL-8

BackgroundPaclitaxel (PTX) is a potent anti-cancer drug commonly used for the treatment of advanced breast cancer (BCA) and melanoma. Toll-like receptor 4 (TLR4) promotes the production of pro-inflammatory cytokines associated with cancer chemoresistance. This study aims to explore the effect of TLR4 in PTX resistance in triple-negative BCA and advanced melanoma and the effect of compound A (CpdA) to attenuate this resistance.MethodsBCA and melanoma cell lines were checked for the response to PTX by cytotoxic assay. The response to PTX of TLR4-transient knockdown cells by siRNA transfection was evaluated compared to the control cells. Levels of pro-inflammatory cytokines, IL-6 and IL-8, and anti-apoptotic protein, XIAP were measured by real-time PCR whereas the secreted IL-8 was quantitated by ELISA in TLR4-transient knockdown cancer cells with or without CpdA treatment. The apoptotic cells after adding PTX alone or in combination with CpdA were detected by caspase-3/7 assay.ResultsPTX could markedly induce TLR4 expression in both MDA-MB-231 BCA and MDA-MB-435 melanoma cell lines having a basal level of TLR4 whereas no significant induction in TLR4-transient knockdown cells occurred. The siTLR4-treated BCA cells revealed more dead cells after PTX treatment than that of mock control cells. IL-6, IL-8 and XIAP showed increased expressions in PTX-treated cells and this over-production effect was inhibited in TLR4-transient knockdown cells. Apoptotic cells were detected higher when PTX and CpdA were combined than PTX treatment alone. Isobologram exhibited the synergistic effect of CpdA and PTX. CpdA could significantly decrease expressions of IL-6, XIAP and IL-8, as well as excreted IL-8 levels together with reduced cancer viability after PTX treatment.ConclusionsThe acquired TLR4-mediated PTX resistance in BCA and melanoma is explained partly by the paracrine effect of IL-6 and IL-8 released into the tumor microenvironment and over-production of anti-apoptotic protein, XIAP, in BCA cells and importantly CpdA could reduce this effect and sensitize PTX-induced apoptosis in a synergistic manner. In conclusion, the possible impact of TLR4-dependent signaling pathway in PTX resistance in BCA and melanoma is proposed and using PTX in combination with CpdA may attenuate TLR4-mediated PTX resistance in the treatment of the patients.

1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) reduces hepatic injury in concanavalin A-treated mice.

1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG), a chemically synthesized monoacetyldiaglyceride, is one of the constituents in Sika deer antlers and has been known traditionally as having immunomodulatory effects. However, the mechanism by which PLAG controls neutrophil migration, which evokes liver injury in the hepatitis animal model, remains largely unknown. This study was designed to evaluate the immunomodulatory effects of PLAG on cytokine secretion and neutrophil migration in vivo and in vitro. Concanavalin A (Con A) induced leukocyte infiltration in the liver and increased plasma cytokine levels. Pretreatment with PLAG reduced the levels of interleukin (IL)-4, IL-6, IL-10, and CXCL2, but maintained interferon (IFN)-γ levels and modulated neutrophil recruitment toward the liver. Furthermore, the mRNA and protein levels of IL-4 and CXCL2 in liver tissue were also decreased in the Con A-treated mice. Liver histology analyses showed that PLAG reduced Con A-induced hepatic necrosis, which was accompanied by leukocyte infiltration. The in vitro studies revealed that PLAG reduced IL-4 secretion in Con A stimulated T cell and blocked signal transducer and activator of transcription 6 (STAT6) Con A induced hepatocyte. PLAG attenuated IL-4 induced activation of atypical protein kinase C (PKC)/STAT6 in hepatocytes and inhibited neutrophil migration toward the liver tissue through suppression of IL-8/vascular cell adhesion molecule (VCAM) expression. These results suggest that PLAG could mitigate excess neutrophil migration into liver tissue and potentially have a therapeutic effect on immune-mediated liver injury.

Host Defense Peptide Expression in Human Cervical Cells and Regulation by 1,25-Dihydroxyvitamin D3 in the Presence of Cytokines and Bacterial Endotoxin.

Host defense peptides (HDPs) in the pregnant female reproductive tract provide protection against infection. The relationship between HDPs and infection/inflammation is poorly understood. Therefore, we investigated the regulation of HDPs by 1α, 25-dihydroxyvitamin D3 (1,25-(OH)2) in the presence/absence of infectious/inflammatory agents. Endocervical epithelial cells (END1/E6E7, n = 6) were exposed to 1,25-(OH)2, calcipotriol, interleukin 1β (IL-1β), granulate-macrophage colony-stimulating factor (GM-GSF), and lipopolysaccharide (LPS). Elafin, human beta defensin (hBD2), cathelicidin, secretory leucocyte protease inhibitor, interleukin 8, 1,25-(OH)2 receptor, and toll-like receptor 4 (TLR4) expression was determined using quantitative polymerase chain reaction and/or enzyme-linked immunosorbent assay. Host defense peptide gene and protein expression was assessed in cervicovaginal cells/fluid, respectively, from first trimester pregnant women (n = 8-12). Interleukin 1β induced elafin and hBD2. The 1,25-(OH)2 induced cathelicidin expression in the presence of IL-1β and LPS. The 1,25-(OH)2 also attenuated IL-1β-induced IL-8 expression and LPS enhancement of TLR4. Host defense peptides and TLR4 profiles in cervicovaginal cells and fluid samples from pregnant women were similar to END1/E6E7 cells. In conclusion, HDPs are differentially regulated in END1/E6E7 cells. The 1,25-(OH)2 induction of cathelicidin and suppression of IL-8 highlights a mechanism by which 1,25-(OH)2 supplementation could enhance the pregnant innate immune defenses.

IFN-gamma priming of adipose-derived stromal cells at "physiological" hypoxia.

Multipotent mesenchymal stromal cells (MSCs) are considered cue regulators of tissue remodeling. Their activity is strongly governed by local milieu, where O2 level is most important. The elevation of inflammatory mediators and acute O2 lowering may additionally modulate MSC activity. In present paper the priming effects of IFN-gamma on adipose tissue-derived MSCs (ASCs) at tissue-related O2 level (5%) and acute hypoxic stress (0.1% O2 ) were assessed as alterations of ASCs' CFU-F, proliferation, migration, osteo-commitment. IFN-gamma priming provoked ROS elevation, cell growth slowdown, attenuation of both spontaneous and induced osteodifferentiation of tissue O2 -adapted ASCs. The prominent changes in ASC cytoskeleton-related gene transcription was detected. IFN-gamma exposure shifted the ASC paracrine profile, suppressing the production of VEGF and IL-8, while MCP-1 and IL-6 were stimulated. Conditioned medium of IFN-gamma-primed ASCs did not activate vessel growth in the CAM assay, but induced endothelial cell migration in "wound closure." Short-term hypoxia suppressed CFU-F number, IFN-gamma-induced elevation of IL-6 and endothelial cell migration, while it abolished IFN-gamma-provoked VEGF inhibition. After N-acetyl cysteine treatment ROS level was partly abolished providing additional enhancement of IL-6 and suppression of IL-8 and VEGF production. These findings demonstrated that paracrine activity of ASCs in part may be governed by ROS level. Thus, this study first demonstrated that IFN-gamma priming itself and in combination with acute O2 deprivation could supply dual effects on ASC functions providing both stimulatory and hampering effects. The equilibrium of these factors is a substantial requirement for the execution of MSC remodeling functions.

Noncanonical NF-\u03baB mediates the Suppressive Effect of Neutrophil Elastase on IL-8/CXCL8 by Inducing NKRF in Human Airway Smooth Muscle

Neutrophil elastase (NE) suppresses IL-8/CXCL8 in human airway smooth muscle cells (hASM) while stimulating its production in respiratory epithelial cells. This differential effect is mediated by the selective induction of NKRF and dysregulation in chronic inflammatory diseases. We hypothesized that the differential activation of NF-κB subunits confer the opposite effect of NKRF on IL-8/CXCL8 in primary hASM and A549 cells stimulated with NE. The events occurring at the promoters of NKRF and IL-8/CXCL8 were observed by ChIP assays, and the functional role of RelB was confirmed by knockdown and overexpression. Although p65 was stimulated in both cell types, RelB was only activated in NE-treated hASM, as confirmed by NF-κB DNA binding ELISA, Western blotting and confocal microscopy. Knockdown of RelB abolished the induction of NKRF and converted the suppression of IL-8/CXCL8 to stimulation. The forced expression of RelB induced NKRF production in hASM and A549 cells. NE activated the NIK/IKK1/RelB non-canonical NF-κB pathway in hASM but not in A549. The nuclear-translocated RelB was recruited to the NKRF promoter around the putative κB site, accompanied by p52 and RNA polymerase II. In conclusion, NFRF is a novel RelB-response gene, and NE is a stimulator of the non-canonical RelB/NF-κB pathway in hASM.

Resveratrol Protects against Helicobacter pylori-Associated Gastritis by Combating Oxidative Stress.

Helicobacter pylori (H. pylori)-induced oxidative stress has been shown to play a very important role in the inflammation of the gastric mucosa and increases the risk of developing gastric cancer. Resveratrol has many biological functions and activities, including antioxidant and anti-inflammatory effect. The purpose of this study was to probe whether resveratrol inhibits H. pylori-induced gastric inflammation and to elucidate the underlying mechanisms of any effect in mice. A mouse model of H. pylori infection was established via oral inoculation with H. pylori. After one week, mice were administered resveratrol (100 mg/kg body weight/day) orally for six weeks. The mRNA and protein levels of iNOS and IL-8 were assessed using RT-PCR, Western blot and ELISA. The expression levels of IκBα and phosphorylated IκBα (which embodies the level and activation of NF-κB), Heme Oxygenase-1 (HO-1; a potent antioxidant enzyme) and nuclear factor-erythroid 2 related factor 2 (Nrf2) were determined using Western blot, and lipid peroxide (LPO) level and myeloperoxidase (MPO) activity were examined using an MPO colorimetric activity assay, thiobarbituric acid reaction, and histological-grade using HE staining of the gastric mucosa. The results showed that resveratrol improved the histological infiltration score and decreased LPO level and MPO activity in the gastric mucosa. Resveratrol down-regulated the H. pylori-induced mRNA transcription and protein expression levels of IL-8 and iNOS, suppressed H. pylori-induced phosphorylation of IκBα, and increased the levels of HO-1 and Nrf2. In conclusion, resveratrol treatment exerted significant effects against oxidative stress and inflammation in H. pylori-infected mucosa through the suppression of IL-8, iNOS, and NF-κB, and moreover through the activation of the Nrf2/HO-1 pathway.

Abstract POSTER-TECH-1106: Rational design of molecularly targeted therapy for GAB2-driven ovarian cancers

Abstract Studies of cancer genomes indicate that high-grade serous ovarian cancers, the most aggressive subtype accounting for over 70% of all ovarian cancer deaths, are characterized by widespread genomic regions of copy number alterations. Our previous study that systematically assessed 455 amplified genes using open reading frame-based expression approaches for their abilities to transform immortalized human cells has identified the signaling adaptor GAB2 (GRB2-associated binding protein 2) as a potent tumor-promoting gene (*Dunn,*Cheung et al, PNAS 2014). GAB2 gene is located at the chromosome 11q14.1 region that is amplified in approximately 38% of primary ovarian tumors and the amplification event has been associated with poor prognosis in patients. This study investigates the signaling mechanisms by which GAB2 amplification promotes ovarian tumor growth. This will provide us insights into the design of a rational therapeutic strategy. Experimental procedures: We established immortalized human fallopian tube secretory epithelial (FTSE) cells that stably overexpressed GAB2 or a control vector. Gene expression profiling and cytokine arrays were applied to identify signaling pathways altered by GAB2 overexpression. The biological roles of these signaling molecules were assessed in human ovarian cancer cell lines harboring GAB2 amplification/overexpression using a combination of cell-based and in vivo approaches. Summary of data: Increased TNFa signaling was observed in GAB2-overexpressing FTSE cells compared to cells expressing a control vector. This resulted in IKKb-dependent induction of multiple cytokines/chemokines, including IL8, CXCL1 and CXCL2 and others. Suppression of IL8 or CXCL1/2 individually significantly inhibited anchorage-independent growth of GAB2-overexpressing FTSE cells. Suppression of GAB2 in ovarian cancer cells also led to downregulation of IL8, CXCL1 and CXCL2 mRNAs and significantly impaired ovarian tumor growth. We are currently testing multiple IKKb small molecule inhibitors for the ability to suppress IL8 and CXCL1/2 expression. We also plan to evaluate their efficacies alone or in combination with chemotherapy in inhibiting tumor angiogenesis and tumor growth in GAB2-amplified tumors. Conclusions: Overexpression of GAB2 promotes IKKb-dependent induction of multiple cytokines that may enhance tumor angiogenesis and growth of ovarian cancers. We are currently evaluating several IKKb small molecule inhibitors as potential agents to increase the therapeutic efficacy of conventional chemotherapy for ovarian cancers. Funding support: Pilot Study Award, Marsha Rivkin Center for Ovarian Cancer Research (H.W.C.), The V Foundation for Cancer Research (H.W.C.), Liz Tiberis Early Career Award, Ovarian Cancer Research Fund (H.W.C.), American Cancer Society Institutional Citation Format: Hiu Wing Cheung, Chris Duckworth. Rational design of molecularly targeted therapy for GAB2-driven ovarian cancers [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-TECH-1106.

IL-8 confers resistance to EGFR inhibitors by inducing stem cell properties in lung cancer.

Epidermal growth factor receptor (EGFR)-targeted strategy is limited by resistance. We identify the potential genes involved in EGFR TKI (tyrosine kinase inhibitor) resistance and study the therapeutic mechanism in the non-small cell lung cancers. Potential genes involved in resistance were examined by analyzing datasets from a pair of EGFR TKI-sensitive (PC9) and TKI-resistant cells (PC9/gef). Blood specimens from patients taking EGFR TKI as first-line treatment were used to examine the correlation between drug's efficacy and IL-8 level. The effects of IL-8 on gefitinib-induced apoptosis, stemness, and in vivo tumorigenicity were investigated using established cell lines. We identified IL-8 was up-regulated in gefitinib-resistant cells, and high plasma IL-8 level was correlated with shorter progression-free-survival time. IL-8 overexpression suppressed gefitinib-induced apoptosis in gefitinib-sensitive cells. By contrast, suppression of IL-8 enhanced gefitinib-induced cell death in gefitinib-resistant cells. IL-8 also increased stem-like characteristics including aldehyde dehydrogenase activity, expression of stemness-related genes, clonogenic activity, side-population, and in vivo tumorigenicity. Consistently, knockdown of IL-8 leads to loss of stem cell-like characteristics in gefitinib-resistant cells. Our study demonstrates an important role for IL-8, and suggests IL-8 is a potential therapeutic target for overcoming EGFR TKI resistance.

Abstract 168: Understanding the role of the carcinoid associated fibroblasts in the neuroendocrine tumor microenvironment

Abstract A lack of cell lines and animal models has been an obstacle in developing novel therapeutic agents for advanced carcinoid tumors. In the past two years we have developed a collection of short-term carcinoid cell lines. Carcinoid cells in culture grow slowly and are importantly attached to, much faster, growing stromal fibroblasts, referred to as CAF's (Carcinoid Associated Fibroblasts). This phenotype is conserved in vivo and closely mimics the status of these tumors in humans. CAF's are known to have pro-tumorigenic effects; are attractive targets for cancer therapy; as well as valuable clinical biomarkers of prognosis. With the availability of an in-house panel of primary cell lines and control cultured fibroblasts, teasing apart the tumor-fibroblast relationship is feasible through collection of cell culture supernatants and in vitro screening of secreted proteins. We present here our initial screening studies using three cell culture environments, namely normal human fibroblasts, carcinoid-associated fibroblasts (CAF's) and the tumor microenvironment co-culture of stromal cells and CAF's. We screened 55 secreted proteins with known carcinogenic associations by multiplexed bead-based (BioPlex, BioRad) analysis. We have identified a potential 5-marker CAF signature encompassing elevated secretion of IL-6 and MCP-1; and the suppression of IL-8 and FGF basic, coupled with VEGF expression. As part of the next phase we plan to utilize our collection of banked whole blood, serum, and plasma specimens comprising over 1000 cases of patients with neuroendocrine tumors, to validate our secreted protein signature in early diagnosis and / or assessing progression and prognosis in patients with neuroendocrine tumors based on stage and tumor subtype. Citation Format: Michaela Bowden, Ewa Sicinska, Matthew Kulke, Massimo Loda. Understanding the role of the carcinoid associated fibroblasts in the neuroendocrine tumor microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 168. doi:10.1158/1538-7445.AM2014-168

Inflammatory Cytokines of Cystic Fibrosis Airway Cells Are Reduced with Exposure to Interleukin-10

Interleukin (IL) 10 deficiency is notable in cystic fibrosis (CF) bronchoalveolar lavage (BAL) fluid. Similar airway neutrophils did not inhibit IL-8 production with IL-10 exposure. This study describes the complex response of CF airway immune cells to recombinant IL-10 exposure when compared to non-CF patients. BAL fluid was obtained from 11 CF and 3 non-CF pediatric subjects and immediately centrifuged at 1,000 rpm for 10 min. The cells were resuspended to a final concentration of 1×106 cells/mL. Aliquots of the cells were incubated with 0, 10, 100, and 1,000 ng/mL of recombinant human IL-10 for 24 h. Supernatants were obtained and enzyme-linked immunosorbent assay performed for levels of IL-8, IL-1β, and tumor necrosis factor alpha (TNFα) cytokines. CF patients (n=11) showed statistically significant percent inhibition of IL-8 at IL-10 level at 10 ng/mL (34.7%, P<0.001) and 100 ng/mL (45.9%, P=0.001), though no statistically significant inhibition noted at 1,000 ng/mL. IL-1β did not show statistically significant percent inhibition at any concentration. TNFα revealed a percent inhibition at IL-10 level of 10 ng/mL (52.4%, P=0.002) and 100 ng/mL (61.0%, P<0.001) and no statistically significant inhibition at 1,000 ng/mL. CF and controls (n=3) were marginally different for IL-1β, but showed no statistically significant difference in percent inhibition for IL-8 and TNFα. CF airway immune cell cytokines demonstrate a similar response to IL-10 as non-CF controls with notable differential suppression of IL-8 and TNFα, though not IL-1β. IL-10 resistant airway cells are less likely to be the cause of CF pulmonary hyperinflammation.

Active targeting of RGD-conjugated bioreducible polymer for delivery of oncolytic adenovirus expressing shRNA against IL-8 mRNA

Even though oncolytic adenovirus (Ad) has been highlighted in the field of cancer gene therapy, transductional targeting and immune privilege still remain difficult challenges. The recent reports have noted the increasing tendency of adenoviral surface shielding with polymer to overcome the limits of its practical application. We previously reported the potential of the biodegradable polymer, poly(CBA-DAH) (CD) as a promising candidate for efficient gene delivery. To endow the selective-targeting moiety of tumor vasculature to CD, cRGDfC well-known as a ligand for cell-surface integrins on tumor endothelium was conjugated to CD using hetero-bifunctional cross-linker SM (PEG) n. The cytopathic effects of oncolytic Ad coated with the polymers were much more enhanced dose-dependently when compared with that of naked Ad in cancer cells selectively. Above all, the most potent oncolytic effect was assessed with the treatment of Ad/CD-PEG 500-RGD in all cancer cells. The enhanced cytopathic effect of Ad/RGD-conjugated polymer was specifically inhibited by blocking antibodies to integrins, but not by blocking antibody to CAR. HT1080 cells treated with Ad/CD-PEG 500-RGD showed strong induction of apoptosis and suppression of IL-8 and VEGF expression as well. These results suggest that RGD-conjugated bioreducible polymer might be used to deliver oncolytic Ad safely and efficiently for tumor therapy.

A Novel Isoform of Microphthalmia-Associated Transcription Factor Inhibits IL-8 Gene Expression in Human Cervical Stromal Cells

Cervical ripening during pregnancy is a profound change in cervix structure and function characterized by increases in the proinflammatory cytokine IL-8 and dissolution of the cervical extracellular matrix. Relatively little is known about the molecular mechanisms that underlie these events. Here, we report identification of a novel isoform of micropthalmia-associated transcription factor in human cervical stromal cells (MiTF-CX) that is down-regulated 12-fold during cervical ripening and that represses expression of IL-8. Ectopic expression of MiTF-CX in human cervical stromal cells resulted in substantial suppression of endogenous IL-8 mRNA and protein expression, whereas expression of dominant negative MiTF-CX mutants with impaired DNA binding resulted in dramatic increases in IL-8 production. Gel shift, reporter gene, and chromatin immunoprecipitation assays revealed one strong binding site (E-box (-397) CACATG(-391)) in the human IL-8 promoter that was crucial for mediating transcriptional repression by MiTF-CX. Moreover, we show that MiTF-CX expression in the cervix was itself positively autoregulated via two E-box motifs within a 2.1-kb promoter fragment. We therefore propose that maintenance of cervical competency during pregnancy is an active process maintained through suppression of IL-8 by the transcription factor MiTF-CX. During cervical ripening, loss of MiTF-CX would result in significant up-regulation of IL-8 mRNA and protein synthesis, thereby leading to recruitment and activation of leukocytes within the cervix and dissolution of the extracellular matrix.