NF-κB Inhibitor Parthenolide Research Articles

Inhibition of reactive oxygen species generation by N-Acetyl Cysteine can mitigate male germ cell toxicity induced by bisphenol analogs

The estrogen-like effect of bisphenol A (BPA) disrupting the maintenance of functional male germ cells is associated with male sub-fertility. This study investigated toxicity of male germ cells induced by four bisphenol analogs: BPA, BPAF, BPF, and BPS. The investigation of bisphenol analogs’ impact on male germ cells included assessing proliferation, apoptosis induction, and the capacity to generate reactive oxygen species (ROS) in GC-1 spermatogonia (spg) cells, specifically type B spermatogonia. Additionally, the therapeutic potential and protective effects of N-Acetyl Cysteine (NAC) and NF-κB inhibitor parthenolide was evaluated. In comparison to BPA, BPF and BPS, BPAF exhibited the most pronounced adverse effect in GC-1 spg cell proliferation. This effect was characterized by pronounced inhibition of phosphorylation of PI3K, AKT, and mTOR, along with increased release of cytochrome c and subsequent cleavages of caspase 3, caspase 7, and poly (ADP-ribose) polymerase. Both NAC and parthenolide were effective reducing cellular ROS induced by BPAF. However, only NAC demonstrated a substantial recovery in proliferation, accompanied by a significant reduction in cytochrome c release and cleaved PARP. These results suggest that NAC supplementation may play an effective therapeutic role in countering germ cell toxicity induced by environmental pollutants with robust oxidative stress-generating capacity.

NF-κB p65 Attenuates Cardiomyocyte PGC-1α Expression in Hypoxia.

Hypoxia exerts broad effects on cardiomyocyte function and viability, ranging from altered metabolism and mitochondrial physiology to apoptotic or necrotic cell death. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a key regulator of cardiomyocyte metabolism and mitochondrial function and is down-regulated in hypoxia; however, the underlying mechanism is incompletely resolved. Using primary rat cardiomyocytes coupled with electrophoretic mobility shift and luciferase assays, we report that hypoxia impaired mitochondrial energetics and resulted in an increase in nuclear localization of the Nuclear Factor-κB (NF-κB) p65 subunit, and the association of p65 with the PGC-1α proximal promoter. Tumor necrosis factor α (TNFα), an activator of NF-κB signaling, similarly reduced PGC-1α expression and p65 binding to the PGC-1α promoter in a dose-dependent manner, and TNFα-mediated down-regulation of PGC-1α expression could be reversed by the NF-κB inhibitor parthenolide. RNA-seq analysis revealed that cardiomyocytes isolated from p65 knockout mice exhibited alterations in genes associated with chromatin remodeling. Decreased PGC-1α promoter transactivation by p65 could be partially reversed by the histone deacetylase inhibitor trichostatin A. These results implicate NF-κB signaling, and specifically p65, as a potent inhibitor of PGC-1α expression in cardiac myocyte hypoxia.

NF-κB Inhibitor Parthenolide Promotes Renal Tubules Albumin Uptake in Type 2 Diabetic Nephropathy.

Objective Injured tubular reabsorption is highlighted as one of the causes of increased albuminuria in the early stage of diabetic nephropathy; however, the underlying mechanism has not been fully elucidated. In this study, we aimed to explore whether reducing inflammation and remodeling the insulin signaling pathway could improve albumin uptake of renal tubules. Methods 8-week-old male db/db mice (n=8), a type 2 diabetic nephropathy model, administered with nuclear factor kappa-B (NF-κB) inhibitor parthenolide (PTN, 1 mg/kg) intraperitoneally every other day for 8 weeks, were as the treatment group. Meanwhile, the age-matched male db/m mice (n=5) and db/db mice (n=8) were treated with saline as the control group and type 2 diabetic nephropathy group. When the mice were sacrificed, blood and urine were collected to examine homeostasis model assessment of insulin resistance (HOMA-IR) and urine albumin creatinine ratio, and kidney samples were used to analyze histopathologic changes with periodic acid-Schiff (PAS) staining, NF-κB p65, phosphorylation of AKT (p-AKT), amnionless and cubilin expressions with immunohistochemistry as well as western blot, and the albumin uptake of renal tubules by using immunofluorescence. In addition, HKC cells were divided into the insulin group treated with insulin alone, the TNF-α group treated with insulin and tumor necrosis factor (TNF-α), and the TNF-α+PTN group exposed to PTN, insulin and TNF-α. The levels of albumin uptake and expression levels of NF-κB p65, p-IRS-1/IRS-1, p-AKT/AKT, amnionless and cubilin in HKC cells were measured. Results Compared with the db/db group, the db/db+PTN group demonstrated decreased levels of HOMA-IR (36.83±14.09 vs. 31.07±28.05) and urine albumin creatinine ratio (190.3±7.3 vs. 143.0±97.6 mg/mmol); however, the differences were not statistically significant (P>0.05). Periodic acid-Schiff staining showed PTN could alleviate the glomerular hypertrophy and reduce the matrix in mesangial areas of db/db mice. The renal expression of NF-κB p65 was increased and p-AKT (s473) decreased in the db/db group compared with the db/m group (P<0.05). PTN significantly reduced the renal expression of NF-κB p65 and ameliorated the decline of p-AKT (s473) compared with the db/db group (P<0.05). Compared with the db/m group, the expression of amnionless and cubilin decreased and albumin uptake in tubules were reduced in the db/db group (P<0.05), and PTN could significantly increase the expression of cubilin (P<0.05), and improve albumin uptake in tubules. Insulin promoted albumin uptake and the expression of amnionless and cubilin in HKC cells (P<0.05). TNF-α stimulated the expression of NF-κB p65, increased p-IRS-1 (s307) and reduced p-AKT (s473) in HKC cells (P<0.05). In the TNF-α+PTN group, the expression of NF-κB p65 declined and p-IRS-1 (s307) and p-AKT (s473) were restored, compared with the TNF-α group (P<0.05). The expression of amnionless and cubilin decreased in the TNF-α group (P<0.05), and PTN could significantly increase the expression of cubilin (P<0.05). Conclusions Inflammation caused damage to insulin signaling, which reduced amnionless-cubilin expression and albumin uptake. PTN could reduce inflammation and remodel the impaired insulin signaling pathway, which promoted the expression of cubilin and albumin uptake. Our study can shed light on the role of inflammation in the reduction of albumin uptake of renal tubules in type 2 diabetic nephropathy.

Regulation of PLPP3 gene expression by NF-κB family transcription factors

Lipid phosphate phosphatase 3 (LPP3), encoded by the PLPP3 gene, is an integral membrane enzyme that dephosphorylates phosphate esters of glycero- and sphingophospholipids. Cell surface LPP3 can terminate the signaling actions of bioactive lysophosphatidic acid (LPA) and sphingosine 1 phosphate, which likely explains its role in developmental angiogenesis, vascular injury responses, and cell migration. Heritable variants in the final intron PLPP3 associate with interindividual variability in coronary artery disease risk that may result from disruption of enhancer sequences that normally act in cis to increase expression of the gene. However, the mechanisms regulating PLPP3 expression are not well understood. We show that the human PLPP3 promoter contains three functional NF-κB response elements. All of these are required for maximal induction of PLPP3 promoter activity in reporter assays. The identified sequences recruit RelA and RelB components of the NF-κB transcription complex to chromatin, and these transcription factors bind to the identified target sequences in two different cell types. LPA promotes binding of Rel family transcription factors to the PLPP3 promoter and increases PLPP3 gene expression through mechanisms that are attenuated by an NF-κB inhibitor, LPA receptor antagonists, and inhibitors of phosphoinositide 3 kinase. These findings indicate that up-regulation of PLPP3 during inflammation and atherosclerosis results from canonical activation of the NF-κB signaling cascade to increase PLPP3 expression through nuclear import and binding of RelA and RelB transcription factors to the PLPP3 promoter and suggest a mechanism by which the LPP3 substrate, LPA, can regulate PLPP3 expression.

Inhibition of NF-\u03baB Reduces Renal Inflammation and Expression of PEPCK in Type 2 Diabetic Mice

Renal gluconeogenesis is markedly promoted in patients with type 2 diabetes mellitus (T2DM); however, the underlying mechanism remains largely unknown. Renal gluconeogenesis is found to be negatively regulated by insulin. T2DM is characterized by chronic and subacute inflammation; however, inflammation has been well recognized to induce insulin resistance. Therefore, this study aimed to investigate whether the enhanced renal gluconeogenesis in T2DM was partially due to the renal inflammation-mediated insulin resistance. If so, whether inflammation inhibitor could partially reverse such change. Diabetic db/db mice and db/m mice were used in our study. Typically, diabetic db/db mice were intraperitoneally treated with 1 mg/kg NF-κB inhibitor parthenolide (PTN) or saline as control every other day. Twelve weeks after treatment, animal samples were collected for measurements. Our results suggested that the expression levels of the inflammatory factors and the gluconeogenic rate-limiting enzyme phosphoenolpyruvate carboxykinase (PEPCK) were up-regulated in renal cortex of both db/db mice and T2DM patients. Moreover, reduced insulin signaling, as well as up-regulated expression of downstream genes FOXO1 and PGC-1ɑ, could be detected in renal cortex of db/db mice compared with that of db/m mice. Consistent with our hypothesis, PTN treatment could alleviate renal inflammation and insulin resistance in db/db mice. Moreover, it could also down-regulate the renal expression of PEPCK, indicating that inflammation could be one of the triggers of insulin resistance and the enhanced renal gluconeogenesis in db/db mice. This study can shed light on the role of inflammation in the enhanced renal gluconeogenesis in T2DM, which may yield a novel target for hyperglycemia.

Chronic low dose ethanol induces an aggressive metastatic phenotype in TRAMP mice, which is counteracted by parthenolide.

Despite advances in prostate cancer therapy, dissemination and growth of metastases results in shortened survival. Here we examined the potential anti-cancer effect of the NF-κB inhibitor parthenolide (PTL) and its water soluble analogue dimethylaminoparthenolide (DMAPT) on tumour progression and metastasis in the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model of prostate cancer. Six-week-old male TRAMP mice received PTL (40mg/kg in 10% ethanol/saline), DMAPT (100mg/kg in sterile water), or vehicle controls by oral gavage thrice weekly until palpable tumour formation. DMAPT treatment slowed normal tumour development in TRAMP mice, extending the time-to-palpable prostate tumour by 20%. PTL did not slow overall tumour development, while the ethanol/saline vehicle used to administer PTL unexpectedly induced an aggressive metastatic tumour phenotype. Chronic ethanol/saline vehicle upregulated expression of NF-κB, MMP2, integrin β1, collagen IV, and laminin, and induced vascular basement membrane degradation in primary prostate tumours, as well as increased metastatic spread to the lung and liver. All of these changes were largely prevented by co-administration with PTL. DMAPT (in water) reduced metastasis to below that of water-control. These data suggest that DMAPT has the potential to be used as a cancer preventive and anti-metastatic therapy for prostate cancer. Although low levels of ethanol consumption have not been shown to strongly correlate with prostate cancer epidemiology, these results would support a potential effect of chronic low dose ethanol on metastasis and the TRAMP model provides a useful system in which to further explore the mechanisms involved.

Abstract 1080: Targeting the PI3K-Akt and NF-κB pathways as a combination therapy in blocking prostate cancer progression

Abstract Akt/protein kinase B and transcription factor NF-κB are important signaling molecules and key survival factors involved in the control of cell proliferation, apoptosis and oncogenesis. Although Akt and NF-κB are components of separate signaling pathways, Akt can induce cell survival signal through NF-κB activation. Previous studies from our laboratory have shown constitutive activation of NF-κB/p65 and PI3K-Akt in clinical prostate cancer specimens and in autochthonous mouse model of prostate cancer. In this study we investigated if these two signaling pathways converge and whether dual targeting is effective in blocking disease progression. Using tissue samples obtained during transurethral prostatic resection and paraffin-embedded sections of benign and malignant prostate tissue, we first analyzed nuclear levels of NF-κB/p65, native/activated Akt levels, and evaluated co-localization of these two proteins. Compared to benign tissue, cancer specimens exhibited constitutive Akt and NF-κB/p65 activation which was more pronounced in high-grade cancer (Gleason grade 7-10). Immunohistochemical analyses further confirmed a progressive increase in the activated form of Akt and NF-κB/p65 in the nucleus of cancer tissues compared to benign specimens, and exhibited co-localization of these proteins in a subset of aggressive cancer. Utilizing androgen-responsive prostate cancer LNCaP cells, with increased Akt activity, and androgen-refractory PC-3 cells harboring high levels of Akt and NF-κB. Individual treatment of cells with Akt Inhibitor VIII (0.075-1.8µM) and NF-κB inhibitor Parthenolide (0.32-60µM) for 24-72 h demonstrated partial suppressive effect in cell growth. Strikingly, concurrent blocking of Akt and NF-κB/p65 at 1:10 molar ratio resulted in potentiated toxicity with marked inhibition in proliferation and induction of cell cycle arrest. Interruption of Akt activation using dominant-negative approach resulted in significant inhibition in Akt-phosphorylation (Ser473) and induction of apoptosis in LNCaP cells, whereas ectopic expression of NF-κB/p65 increased doubling time and invasiveness in these cells, which was abrogated by combinational treatment. Furthermore, combination treatment decreased the expression of p-IKK (Ser176/180), p-NF-κB/p65 (Ser536), and p-Akt (Ser473) and downstream targets viz. cyclin D1, Bcl-2, VEGF and MMP9 in both cell lines. In vivo administration of Inhibitor VIII and Parthenolide at 1:10 ratio to PC-3 tumor xenograft for 8 weeks resulted in marked decrease in tumor volume and reduced expression of target genes, compared to individual treatments. These results suggest that nuclear co-localization of Akt and NF-κB/p65 may be developed as prognostic biomarker and concurrent targeting of these molecules by combination of pharmacological inhibitors is an effective approach and is more efficacious than use of single agent blocking prostate cancer progression. Citation Format: Eswar Shankar, Rajnee Kanwal, Aditi Goel, Xiaoping Yang, Sanjeev Shukla, Gregory T. MacLennan, Pingfu Fu, Anant Madabhushi, Parameswaran Ramakrishnan, Sanjay Gupta. Targeting the PI3K-Akt and NF-κB pathways as a combination therapy in blocking prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1080. doi:10.1158/1538-7445.AM2017-1080

PD33-02 PROSTATE CANCER AGGRESSIVENESS IS MEDIATED BY AKT AND NF-κB SIGNALING PATHWAYS: A SYSTEMS BIOLOGY APPROACH

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I1 Apr 2017PD33-02 PROSTATE CANCER AGGRESSIVENESS IS MEDIATED BY AKT AND NF-κB SIGNALING PATHWAYS: A SYSTEMS BIOLOGY APPROACH Eswar Shankar, Rajnee Kanwal, Aditi Goel, Xiaoping Yang, Sanjeev Shukla, Gregory MacLennan, Pingfu Fu, Jing Li, Anant Madabhushi, and Sanjay Gupta Eswar ShankarEswar Shankar More articles by this author , Rajnee KanwalRajnee Kanwal More articles by this author , Aditi GoelAditi Goel More articles by this author , Xiaoping YangXiaoping Yang More articles by this author , Sanjeev ShuklaSanjeev Shukla More articles by this author , Gregory MacLennanGregory MacLennan More articles by this author , Pingfu FuPingfu Fu More articles by this author , Jing LiJing Li More articles by this author , Anant MadabhushiAnant Madabhushi More articles by this author , and Sanjay GuptaSanjay Gupta More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.3316AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prostate cancer remains the second leading cause of cancer-related deaths in American men. Often an indolent disease, patients remain asymptomatic for years. Easily treatable tumors and other, often fatal, highly aggressive forms are difficult to distinguish. Current prognosis and treatment stratification do not accurately predict clinical outcome. Hence there is an urgent need for improved markers to determine prognosis and appropriate treatments. We identified two key signaling pathways (PI3K-Akt and NF-κB) whose constitutive activation correlates with prostate cancer progression. Using an integrated approach of quantitative experimentation and mathematical modeling, we seek to develop a multi-level, hierarchical, quantitative systems biology model - where the lower level captures the dynamic molecular processes of the signaling pathways and the higher level models the cancer phenotype. METHODS p-Akt (Ser473) and NF-κB/p65 protein expression was analyzed in benign and cancer specimens of various Gleason grades and their co-localization by immunohistochemistry. We also utilized androgen-responsive LNCaP cells (possessing increased Akt activity due to mutation in PTEN gene) and androgen-refractory PC-3 cells with constitutive activation of Akt and NF-κB and their treatment with specific inhibitors. Western blotting was performed to determine the expression of native/active forms of Akt and NF-κB and their effector proteins. Subsequent work include connecting the model of cell signaling to the physiologically ″higher″ levels using mechanistic details or statistical models. RESULTS Compared to benign tissue, cancer specimens exhibited constitutive activation of p-Akt (Ser473) and NF-κB/p65 which was more pronounced in high-grade cancer (Gleason grade 7-10). Immunohistochemical analyses further demonstrated co-localization of these proteins in a subset of aggressive cancer tissues. Individual treatment of cell lines with Akt Inhibitor VIII (0.075-1.8μM) and NF-κB inhibitor Parthenolide (0.32-60μM) for 24-72 h demonstrated partial suppressive effect in cancer cell growth; whereas concurrent blocking of Akt and NF-κB/p65 at 1:10μM ratio resulted in potentiated toxicity and inhibition of downstream effector proteins in both cell lines. The categorical behaviors were explored by extensive simulations conducted to explore possible parameterizations of the model and to define the range of potential responses. A differential equation based (mass action and Michaelis-Menten) mathematical model was constructed and structurally calibrated/validated using experimental data. The resulting behaviors were identified and categorized according to the severity of pathway activity. Model parameterization (numerical values of reaction rates and basal protein levels) was accomplished by adopting portions of the models estimating the remaining unknowns from available experimental data. CONCLUSIONS Our results suggest that Akt activation provides long-term cell survival by activating pathways that influence NF-κB-dependent gene transcription, and hence plays a role in prostate cancer aggression. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e594 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Eswar Shankar More articles by this author Rajnee Kanwal More articles by this author Aditi Goel More articles by this author Xiaoping Yang More articles by this author Sanjeev Shukla More articles by this author Gregory MacLennan More articles by this author Pingfu Fu More articles by this author Jing Li More articles by this author Anant Madabhushi More articles by this author Sanjay Gupta More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract P3-04-12: Both spliced and unspliced XBP1 regulates breast cancer cell fate response to antiestrogen via NFkappaB signaling

Abstract Unfolded protein response (UPR), a stress-induced survival mechanism, may be used by cancer cells to avoid cell death. Antiestrogen therapy, widely applied in the treatment of estrogen receptor-positive (ER+) breast cancer, induces endoplasmic reticulum stress (EnR stress) that leads to activation of each of the three arms of the UPR. One critical prosurvival activator that is regulated by two arms of the UPR is the transcription factor X-box binding protein 1 (XBP1). XBP1 exists in two isoforms, the transcriptionally inactive unspliced XBP1(U) and the spliced, active XBP1(S). Overexpression of XBP1(S) confers estrogen independence and antiestrogen resistance in ER+ breast cancer cells and XBP1(S) expression correlates with poor clinical responsiveness to Tamoxifen in ER+ breast tumors. However, the underlying signaling mechanisms regulated by XBP1, which may mediate its effects on antiestrogen resistance, are unknown. We show that depletion of endogenous XBP-1 by siRNA increases apoptosis, decreases autophagy, requires down-regulation of p65/RelA, a component of the pro-survival NFκB complex. Using novel spliced and non-spliceable forms of XBP1, we show that XBP1(U) and XBP1(S) both regulate NFκB activity via ERα signaling in breast cancer cells. XBP1(S), but not XBP1(U), also can regulate p65/RelA expression independent of ERα. Antiestrogen resistance as conferred by XBP1 overexpression in MCF-7 cells requires the activation of NFκB signaling; inhibition of NFκB signaling by either the small molecule NFκB inhibitor Parthenolide or p65/RelA siRNA sensitizes XBP1 overexpressing cells to Tamoxifen. The activation of XBP1 and the downstream NFκB signaling is likely to contribute to the TMEM33 overepxression induced apoptosis, as both NFκB signaling and XBP1(S) are elevated in TMEM33 overexpressed MCF7 cells. Thus, we have identified a critical regulatory link between the UPR/XBP1 pathway and pro-survival NFκB signaling that is a major contributor to endocrine responsiveness in ER positive breast cancer. Citation Format: Clarke R, Hu R, Warri A, Jin L, Zwart A, Riggins R, Fang H-b. Both spliced and unspliced XBP1 regulates breast cancer cell fate response to antiestrogen via NFkappaB signaling [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-12.

The clerodane diterpene casearin J induces apoptosis of T-ALL cells through SERCA inhibition, oxidative stress, and interference with Notch1 signaling.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that preferentially affects children and adolescents. Over 50% of human T-ALLs possess activating mutations of Notch1. The clerodane diterpene casearin J (CJ) is a natural product that inhibits the sarcoendoplasmatic reticulum calcium ATPase (SERCA) pump and induces cell death in leukemia cells, but the molecular mechanism of cytotoxicity remains poorly understood. Here we show that owing to SERCA pump inhibition, CJ induces depletion of the endoplasmic reticulum calcium pools, oxidative stress, and apoptosis via the intrinsic signaling pathway. Moreover, Notch1 signaling is reduced in T-ALL cells with auto-activating mutations in the HD-domain of Notch1, but not in cells that do not depend on Notch1 signaling. CJ also provoked a slight activation of NF-κB, and consistent with this notion a combined treatment of CJ and the NF-κB inhibitor parthenolide (Pt) led to a remarkable synergistic cell death in T-ALL cells. Altogether, our data support the concept that inhibition of the SERCA pump may be a novel strategy for the treatment of T-ALL with HD-domain-mutant Notch1 receptors and that additional treatment with the NF-κB inhibitor parthenolide may have further therapeutic benefits.

The HIV proteins Tat and Nef promote human bone marrow mesenchymal stem cell senescence and alter osteoblastic differentiation.

To maintain bone mass turnover and bone mineral density (BMD), bone marrow (BM) mesenchymal stem cells (MSCs) are constantly recruited and subsequently differentiated into osteoblasts. HIV-infected patients present lower BMD than non-HIV infected individuals and a higher prevalence of osteopenia/osteoporosis. In antiretroviral treatment (ART)-naive patients, encoded HIV proteins represent pathogenic candidates. They are released by infected cells within BM and can impact on neighbouring cells. In this study, we tested whether HIV proteins Tat and/or Nef could induce senescence of human BM-MSCs and reduce their capacity to differentiate into osteoblasts. When compared to nontreated cells, MSCs chronically treated with Tat and/or Nef up to 30 days reduced their proliferative activity and underwent early senescence, associated with increased oxidative stress and mitochondrial dysfunction. The antioxidant molecule N-acetyl- cysteine had no or minimal effects on Tat- or Nef-induced senescence. Tat but not Nef induced an early increase in NF-κB activity and cytokine/chemokine secretion. Tat-induced effects were prevented by the NF-κB inhibitor parthenolide, indicating that Tat triggered senescence via NF-κB activation leading to oxidative stress. Otherwise, Nef- but not Tat-treated cells displayed early inhibition of autophagy. Rapamycin, an autophagy inducer, reversed Nef-induced senescence and oxidative stress. Moreover, Tat+Nef had cumulative effects. Finally, Tat and/or Nef decreased the MSC potential of osteoblastic differentiation. In conclusion, our in vitro data show that Tat and Nef could reduce the number of available precursors by inducing MSC senescence, through either enhanced inflammation or reduced autophagy. These results offer new insights into the pathophysiological mechanisms of decreased BMD in HIV-infected patients.

Fn14 in podocytes and proteinuric kidney disease

Non-proliferative proteinuric diseases are the most common primary glomerular disorders causing end-stage renal disease. These disorders may associate low level glomerular inflammation and podocyte expression of inflammatory mediators. However, the factors regulating podocyte expression of inflammatory mediators in vivo in non-immune disorders are poorly understood. We have now explored the regulation and role of TWEAK receptor Fn14 in mediating glomerular inflammation in cultured podocytes and in experimental and human non-immune proteinuria.Transcriptomics disclosed Fn14 and MCP-1 mRNA upregulation in glomeruli from patients with focal segmental glomerulosclerosis, as well as a correlation between the expression of both transcripts. Increased glomerular Fn14 and MCP-1 mRNA was confirmed in a second focal segmental glomerulosclerosis cohort and was also observed in membranous nephropathy. In human non-proliferative proteinuric kidney diseases podocytes displayed Fn14 and MCP-1 expression and NFκB activation. Podocyte Fn14 was increased in murine protein overload-induced proteinuria. In Fn14 knock-out mice with protein overload-induced proteinuria, glomerular and periglomerular macrophage infiltrates were reduced, as were MCP-1 mRNA and podocyte MCP-1 staining and podocyte numbers preserved as compared to wild-type counterparts. Adenovirus-mediated overexpression of TWEAK increased periglomerular macrophage infiltration in mice without prior kidney injury. In cultured podocytes inflammatory cytokines increased Fn14 mRNA and protein levels. TWEAK activated NFκB and increased MCP-1 mRNA and protein, an effect prevented by the NFκB inhibitor parthenolide. In conclusion, Fn14 activation results in NFκB-mediated pro-inflammatory effects on podocytes that may be relevant for the pathogenesis of non-proliferative proteinuric kidney disease of non-immune origin.

Role of MIF/CXCL8/CXCR2 signaling in the growth of nasopharyngeal carcinoma tumor spheres

Macrophage migration inhibitory factor (MIF) and CXCL8 (also named IL-8) are strongly expressed in the tissues of nasopharyngeal carcinoma (NPC). However, their role in the growth of NPC has not been fully examined. This study aims to evaluate the functions of MIF and CXCL8 on the growth of NPC tumor spheres. The elevated expression of CXCL8 in tumor over normal tissues was confirmed in 37 pairs of biopsies from NPC patients. In the in vitro study, all the poorly differentiated NPC cell lines, including the EBV-positive C666-1, and the EBV-negative CNE-1, CNE-2, SUNE-1, HNE-1 and HONE-1 cells, were found to express CXCL8 and MIF. Therefore, the EBV-positive C666-1 cell was selected to examine for the role of MIF and CXCL8 in the growth of the NPC tumor spheres. Functional study showed that the growth of C666-1 tumor spheres, under the nutrient poor or growth factor supplemented culture conditions, could be inhibited by the CXCL8 specific peptide inhibitor. The growth of the tumor spheres could also be reduced by the CXCR2 specific inhibitor SB225002 or the PI3K/AKT inhibitor LY294002, indicating that the endogenously produced CXCL8 plays an autocrine role in the growth of the tumor spheres. Further mechanistic studies revealed that the gene expression of CXCL8 could be reduced by the MIF specific small interfering RNA (siRNA) or NF-κB inhibitor parthenolide, and the growth of tumor spheres was also reduced after MIF siRNA transfection. Taken together, the present study highlights the role of MIF/CXCL8/CXCR2 axis in the growth of NPC tumor spheres. Chemotherapeutic interference of this signaling pathway may help to control the growth of the NPC tumor.

The role of Toll like receptor 4 pathway in palmitate-induced interleukin-6 expression in human aortic vascular smooth muscle cells

The recombinant adenovirus Toll like receptor 4 (TLR4) shRNA vector (pGSadeno-TLR4) was constructed and transfected into human aortic vascular smooth muscle cells (HA-VSMC).After HA-VSMC were treated with palmitate or different signaling pathway inhibitors,the mRNA and protein levels of interleukin-6 (IL-6)and NF-κB activity were tested with real-time PCR and ELISA,respectively.The results showed that palmitate increased mRNA and protein levels of IL-6 in HA-VSMC in a dose-dependent manner.The expression of IL-6 mRNA reached peak after treatment with 400 μmol/L of palmitate for 6 h,being 10.43 fold of control (P＜0.01).Treatment with 400 pmol/L of palmitate for 24 h maximally upregulated the protein level of IL-6,which was 2.18 fold of control (P＜0.01).NF-κB inhibitor parthenolide markedly inhibited palmitate-stimulated increased in IL-6 mRNA level by 65％ and protein level by 59％ (both P＜0.01).Protein kinase C (PKC) inhibitor chlerythrine suppressed palmitateinduced IL-6 mRNA expression by 24％ and IL-6 protein level by 28％.By contrast,extracellular signal-regulated protein kinase inhibitor PD98059 and phosphatidylinositol 3-kinase inhibitor wortmannin had no effect on the induction of IL-6 by palmitate.Blockade of TLR4 with pGSadeno-TLR4 significantly suppressed palmitate-induced IL-6 mRNA expression by 72％ and IL-6 protein expression by 75％ (both P＜0.01),along with decrease of NF-κB p65 activity decreased by 62％.These results suggest that TLR4/NF-κB and PKC pathways mediate palmitate-induced IL-6 expression in HA-VSMC. Key words: Human aortic vascular smooth muscle cells; Palmitate; Toll like receptor 4 ; Intedeukin-6

The NFκB inhibitor parthenolide reduces cell proliferation and PGE2 synthesis in human endometriotic stromal cells and inhibits development of endometriosis in murine model

Parthenolide is a sesquiterpene lactone derived from the plant feverfew, which has been used in folk medicine for its anti-inflammatory action. We sought to evaluate effects of parthenolide on ESCs, and on endometriotic lesions in a murine model. Aim of this study was to investigate usefulness of parthenolide as a possible therapeutic agent for endometriosis Experimental study. Ectopic endometrial tissue from ovarian chocolate cysts was collected from premenopausal women. ESCs were isolated from these tissues. ESCs were pretreated with parthenolide and exposed to TNFα. After treatment with TNFα, the levels of IL-8 and COX-2 gene expression were evaluated by real-time RT-PCR. After adding TNFα, IL-8 and PGE2 protein expression were determined by ELISA. Cell proliferation after adding TNFα was assessed by BrdU-ELISA. TNFα-induced phosphorylation of signaling pathways was evaluated by Western blot analysis. As an endometriosis model, estradiol-treated ovariectomized mice were injected intraperitoneally with endometrial fragments of donor mice. After 4 weeks of parthenolide injections, the extent of endometriotic lesions was evaluated. With parthenolide pretreatment, TNFα-induced IL-8 gene and protein expression were significantly repressed. TNFα-induced COX-2 expression and PGE2 synthesis were also inhibited. Adding parthenolide significantly suppressed BrdU incorporation of ESCs. Pretreatment with parthenolide inhibited TNFα-induced IκB phosphorylation, although it did not attenuate ERK1/2, p38MAPK, Akt, and JNK1/2 phosphorylation. Using the mice endometriosis model, administrating parthenolide significantly reduced surface area and weight of endometriotic lesions. These findings suggest that parthenolide represses development of endometriosis possibly by suppressing peritoneal inflammatory status through the NFκB pathway.

TWEAK (tumor necrosis factor–like weak inducer of apoptosis) activates CXCL16 expression during renal tubulointerstitial inflammation

TWEAK (tumor necrosis factor-like weak inducer of apoptosis) is a TNF superfamily cytokine that activates the fibroblast growth factor-inducible 14 (Fn14) receptor. Transcriptional analysis of experimental kidney tubulointerstitial inflammation showed a correlation between an upregulation of the mRNA for the transmembrane chemokine CXCL16, a T-cell chemoattractant, and Fn14 activation. Exogenous TWEAK increased mouse kidney CXCL16 expression and T-lymphocyte infiltration in vivo, processes inhibited by the NF-κB inhibitor parthenolide. Tubular cell CXCL16 was increased in a nephrotoxic tubulointerstitial inflammation model and neutralizing anti-TWEAK antibodies decreased this CXCL16 expression and lymphocyte infiltration. In human kidney biopsies with tubulointerstitial inflammation, tubular cell CXCL16 and Fn14 expressions were associated with inflammatory infiltrates. TWEAK upregulated CXCL16 mRNA expression in cultured renal tubular cells in an NF-κB-dependent manner and increased soluble and cellular CXCL16 protein. CXCL16 modestly promoted the expression of cytokines in tubular cells expressing its receptor (CXCR6) and appeared to synergize with TWEAK to promote an inflammatory response; however, it did not modulate tubular cell proliferation or survival. Thus, TWEAK upregulates the expression of the chemokine CXCL16 in tubular epithelium and this may contribute to kidney tubulointerstitial inflammation.

Investigating the Effect of Hsp90 Inhibitors on Initiating Cells in Childhood Acute Lymphoblastic Leukaemia

Abstract 2952Enhanced risk stratification protocols and intensified therapies have improved outcomes and reduced the risk of relapse in childhood acute lymphoblastic leukaemia (ALL). Nevertheless, 20% of patients relapse, due to failure to eradicate the disease. Current chemotherapeutic regimens are designed around the properties of the bulk leukaemia cells, which might differ from those of the leukaemia initiating cell populations (LIC). Therefore, if drugs have no effect on LIC, these cells may expand and eventually cause relapse. Since several populations in childhood ALL have been shown to have LIC properties, developing therapies that are effective against all LIC populations may prevent relapse. We have previously shown that the NF-κB inhibitor parthenolide (PTL) could prevent proliferation and engraftment of unsorted cells and all LIC populations in NSG mice, in most cases examined. Heat shock protein (Hsp) 90 inhibitors are promising targets in cancer therapy. Targeting this protein has a combined impact on many oncogenic pathways involved in malignant progression. In order to investigate whether the ablation of LIC that we observed using PTL could be improved, we examined the effects of two Hsp90 inhibitors on childhood ALL cells in this study. 17-DMAG is used in preclinical and clinical phase I and II testing in breast cancers. It targets the binding site of ATP in Hsp90 and has low toxicity and high oral bioavailability. Celastrol is a novel Hsp90 inhibitor, which has recently been shown to eradicate LIC in AML by inhibiting NF-κB survival signals and inducing oxidative stress. Dose-response effects using 0.01–100nM of each Hsp90 inhibitor were assessed in primary T- and B-ALL cases and on normal haemopoietic cells at 24, 48 and 72h. Cells were stained with Annexin V and PI then viability and apoptosis were assessed by flow cytometry. An IC50 was observed in leukaemia samples using 1nM of both 17-DMAG and Celastrol after 72h. Increasing the dose of 17-DMAG to 100nM and reducing the incubation time to 48 hours for each drug further reduced ALL cell survival, without impacting normal cells. At these doses, 17-DMAG reduced the viability of T-ALL cells to 36±30% and B-ALL cells to 32±13%. In T-ALL cases, 43±15% survived treatment with Celastrol and similar results were observed with B-ALL cells (43±16%). Normal haemopoietic cells were relatively unaffected at these doses (17-DMAG: 81±2% and Celastrol: 86±36%). Subsequently, T-ALL cells were sorted for expression of CD34 and CD7 and B-ALL cells were sorted for CD34 and CD19. The subpopulations were treated with 1nM of each inhibitor and the results compared to those observed using untreated controls. However, at these concentrations the drugs had limited effects on the ALL subpopulations; 31–100% and 28–89% T-ALL subpopulations survived treatment with 17-DMAG and Celastrol respectively. Similar results were observed with B-ALL subpopulations, 9–86% survived treatment with 17-DMAG and 62–100% survived following Celastrol treatment. A number of studies have shown that a regulatory loop may exist between Hsp90 and NF-κB in that downregulation of NF-κB could lead to reduction in Hsp90 protein levels. Therefore, we investigated whether using the Hsp90 inhibitors in combination with a NF-κB inhibitor would be more effective. Samples were treated with 100nM 17-DMAG or 1nM Celastrol for 48h and 7.5mM PTL was added for the last 24 hours. In 3 T-ALL cases, PTL reduced the viability to 28±13%, 17-DMAG to 25±12% and Celastrol to 35±15%. When PTL was used in combination, cell survival was further reduced to only 18±9% (PTL + 17-DMAG) and to 19±10% (PTL + Celastrol). In 3 B-ALL cases, PTL treatment reduced viability to 57±9%, similar results were seen with 17-DMAG (59±6%), while Celastrol appeared to be the most effective of the 3 drugs (38±4%, P=0.02). When PTL was combined with the Hsp90 inhibitors the cell killing was increased by 2 fold compared to PTL or 17-DMAG alone (PTL + 17-DMAG: 31±6%, P=0.04 and PTL + Celastrol: 28±3% P=0.01). Results to date indicate a promising role for the use of Hsp90 inhibitors with PTL and data on the functional ability of treated LIC populations will provide further information on the potential of these drug combinations. In conclusion, these Hsp90 inhibitors were as effective as PTL against childhood leukaemia cells and when used in combination with PTL, cell survival was further reduced by up to 20%. Disclosures:No relevant conflicts of interest to declare.

The Interplay between NF-kappaB and E2F1 Coordinately Regulates Inflammation and Metabolism in Human Cardiac Cells

Pyruvate dehydrogenase kinase 4 (PDK4) inhibition by nuclear factor-κB (NF-κB) is related to a shift towards increased glycolysis during cardiac pathological processes such as cardiac hypertrophy and heart failure. The transcription factors estrogen-related receptor-α (ERRα) and peroxisome proliferator-activated receptor (PPAR) regulate PDK4 expression through the potent transcriptional coactivator PPARγ coactivator-1α (PGC-1α). NF-κB activation in AC16 cardiac cells inhibit ERRα and PPARβ/δ transcriptional activity, resulting in reduced PGC-1α and PDK4 expression, and an enhanced glucose oxidation rate. However, addition of the NF-κB inhibitor parthenolide to these cells prevents the downregulation of PDK4 expression but not ERRα and PPARβ/δ DNA binding activity, thus suggesting that additional transcription factors are regulating PDK4. Interestingly, a recent study has demonstrated that the transcription factor E2F1, which is crucial for cell cycle control, may regulate PDK4 expression. Given that NF-κB may antagonize the transcriptional activity of E2F1 in cardiac myocytes, we sought to study whether inflammatory processes driven by NF-κB can downregulate PDK4 expression in human cardiac AC16 cells through E2F1 inhibition. Protein coimmunoprecipitation indicated that PDK4 downregulation entailed enhanced physical interaction between the p65 subunit of NF-κB and E2F1. Chromatin immunoprecipitation analyses demonstrated that p65 translocation into the nucleus prevented the recruitment of E2F1 to the PDK4 promoter and its subsequent E2F1-dependent gene transcription. Interestingly, the NF-κB inhibitor parthenolide prevented the inhibition of E2F1, while E2F1 overexpression reduced interleukin expression in stimulated cardiac cells. Based on these findings, we propose that NF-κB acts as a molecular switch that regulates E2F1-dependent PDK4 gene transcription.

EGFR and IL-1 Signaling Synergistically Promote Keratinocyte Antimicrobial Defenses in a Differentiation-Dependent Manner

Ligands of the EGF family regulate autocrine keratinocyte proliferation, and IL-1 family cytokines orchestrate epithelial defense responses. Although members of both families are overexpressed in wound healing and psoriasis, their roles in regulating the innate immune functions of keratinocytes remain incompletely explored. Using sensitive assays, we found significant increases of heparin-binding EGF-like growth factor, transforming growth factor-α, and amphiregulin mRNA and protein in lesional psoriasis compared with uninvolved or control skin. In normal human keratinocyte (NHK) monolayers, EGFR ligands were ineffective in inducing DEFB4, S100A7, and CCL20 mRNAs and human β-defensin (hBD)-2 peptide. Combined with IL-1α, however, EGFR ligands provoked 250 × more DEFB4 and CCL20 and a 9-fold rise in S100A7 mRNA relative to the EGFR ligand alone. This synergy was also reflected in secreted hBD-2 protein, both from NHK and reconstituted human epidermis. Keratinocyte differentiation was critical for these responses, as postconfluent NHK yielded mRNA and protein levels an order of magnitude greater than subconfluent cells. Differentiation also influenced signal transduction, with subconfluent cells using NF-κB and postconfluent cells using EGFR, MEK1/2, and p38. We propose that EGFR ligands are important modifiers of IL-1 activity, synergizing with IL-1 to stimulate epidermal production of hBD-2, S100A7, and CCL20, three of the most upregulated transcripts in psoriatic plaques.

Nitroglycerin alters matrix remodeling proteins in THP-1 human macrophages and plasma metalloproteinase activity in rats

Several studies suggested that long-term nitrate therapy may produce negative outcomes in patient mortality and morbidity. A possible mechanism may involve nitrate-mediated activation of various extracellular matrix (ECM) proteases, particularly matrix metalloproteinase-9 (MMP-9), and adhesion molecules in human macrophages, leading to the destabilization of atherosclerotic plaques. We examined the gene and protein regulating effects on THP-1 human macrophages by repeated exposure to therapeutically relevant concentrations of nitroglycerin (NTG) and possible involvement of nuclear factor (NF)-κB signaling mechanism in mediating some of these observed effects. THP-1 human macrophages repeatedly exposed to NTG (at 10 nM, added on days 1, 4 and 7) exhibited extensive alterations in the expression of multiple genes encoding ECM proteases and adhesion molecules. These effects were dissimilar to those produced by a direct nitric oxide donor, diethylenetriamine NONOate. NTG exposure significantly up-regulated NF-κB DNA nuclear binding activity and MMP-9 protein expression, and reduced tissue inhibitor of metalloproteinase-1 (TIMP-1) expression; these effects were abrogated in the presence of the NF-κB inhibitor parthenolide (a chemical inhibitor derived from the feverfew plant). Further, we examined whether our in vitro findings (an elevated MMP-9/TIMP-1 ratio and gelatinase activity) can be translated to in vivo effects, in a rat model. Sprague–Dawley rats exposed continuously to NTG subcutaneously for 8 days via mini-osmotic pumps showed significant induction of plasma MMP-9 dimer concentrations and the expression of a complex of MMP-9 with lipocalin-2 or neutrophil gelatinase associated lipocalin (NGAL). Plasma gelatinase activity was significantly increased by NTG over the entire study period, attaining peak elevation at day 6. Plasma TIMP-1 protein was down-regulated significantly by day 2 and days 4–7 in the NTG-treated rats. Pharmacokinetic monitoring of NTG and its dinitrate metabolites indicated that concentrations were well within therapeutic levels observed in humans. Our studies indicate that clinically relevant concentrations of NTG not only altered ECM matrix by changing the expression of multiple genes that govern cellular integrity, affecting cellular MMP-9/TIMP-1 balance in THP-1 human macrophages possibly via NF-κB activation, but also led to systemic changes in MMP-9/TIMP-1 expression and gelatinase activity in rats. These effects may contribute to extracellular matrix degradation and possible atherosclerotic plaque destabilization.