Inhibition Of NF-kappa Research Articles

Pharmacological interaction between NSAIDS with clomipramine and risperidone in mice visceral pain.

Nonsteroidal anti-inflammatory drugs (NSAIDs) possess as primary action mechanism the inhibition of cyclooxygenases (COX-1, COX-2, and COX-3), thus producing a decreasing prostaglandin synthesis. This study was designed to evaluate whether the antinociception induced by NSAIDs could be modulated by clomipramine or risperidone using a chemical model of inflammatory acute visceral pain, the abdominal acetic acid induced a writhing test in mice. Dose-response curves, intraperitoneal, or intrathecal for the antinociceptive activity displayed by ketoprofen, piroxicam, nimesulide, parecoxib, and paracetamol were analyzed in order to obtain the ED50 of each drug. Pretreatment of mice with either clomipramine or risperidone, increased antinociceptive potency of ketoprofen, piroxicam, nimesulide, parecoxib, and paracetamol, expressed by a decrease in the values of antinociceptive ED50. The results that were obtained are in line with those where the inhibition of COXs provides a justification for most of the pharmacological actions. Nevertheless, several findings suggest other molecular mechanisms, among which may be mentioned, L-selecting shedding; inhibition of i-NOS; inhibition of NF-Kappa B; suppression metaloproteinasas; inhibition of ß2 integrin activation; activation of α2 -adrenoceptor; increase of IL-1ß; upregulation IL-6. In conclusion, the data generated in this study demonstrated that risperidone and clomipramine, separately, increase antinociceptive potency of NSAIDs in a chemical model of inflammatory acute visceral tonic pain.

Abstract CT160: Ixazomib in combination with carboplatin in pretreated women with advanced triple negative breast cancer, an ongoing phase I/II trial (AGMT MBC-10 trial)

Abstract Background: Triple-negative breast cancer (TNBC) comprises a heterogeneous group of diseases which are generally associated with poor prognosis. Recently, the PARP inhibitor olaparib was approved as first targeted treatment beyond antiVEGF therapy for the BRCA1/2 mutated subgroup of TNBC. However, cytotoxic agents still remain the mainstay of treatment for this breast cancer subtype. Ixazomib is a selective and reversible inhibitor of the proteasome, which has been mainly investigated as treatment of multiple myeloma. In a preclinical cell line model for TNBC the first-generation proteasome inhibitor bortezomib showed synergistic efficacy with cisplatin. Clinical data are available for carboplatin plus bortezomib in metastatic ovarian and lung cancers showing remarkable antitumor activity (47% and 38% response rate, respectively) and good tolerability. In solid tumors cytotoxic effect of proteasome inhibitors is thought to be mediated through different mechanisms: (1) Inhibition of the Fanconi Anemia and BRCA1 DNA repair mechanism (2) Inhibition of p53 degradation (3) Inhibition of NF-kappa B signaling cascade. Based on this evidence, the phase I/II MBC-10 trial will evaluate the toxicity profile and efficacy of the oral second-generation proteasome inhibitor ixazomib in combination with carboplatin in patients with advanced TNBC. Trial Design: Patients with metastatic TNBC pretreated with at least one prior line of chemotherapy for advanced disease with a confirmed disease progression and measurable disease are eligible for this study. Patients will receive ixazomib in combination with carboplatin on days 1, 8, and 15 in a 28-day cycle. The phase I part of this study uses an alternate dose escalation accelerated titration design. After establishing the maximum tolerated dose (MTD), accrual continues to evaluate the efficacy and safety of the combination (phase II, including 41 evaluable patients). All patients will continue on study drugs until disease progression, unacceptable toxicity or discontinuation for any other reason. Primary endpoint of the phase II is overall response rate, secondary endpoints include safety profile, progression-free survival and quality of life. The MBC-10 trial is accompanied by a broad biomarker program investigating predictive biomarkers for treatment response and potential resistance mechanisms to the investigational drug combination. This trial is open for patient enrollment since November 2016 in six Austrian cancer centers. Accrual is planned to be completed within two years. ClinicalTrials.gov Identifier: NCT02993094 Citation Format: Gabriel Rinnerthaler, Simon P. Gampenrieder, Andreas Petzer, Renate Pusch, Daniela Voskova, Dieter Rossmann, Marija Balic, Daniel Egle, Holger Rumpold, Christian F. Singer, Thomas Melchardt, Hanno Ulmer, Brigitte Mlineritsch, Richard Greil. Ixazomib in combination with carboplatin in pretreated women with advanced triple negative breast cancer, an ongoing phase I/II trial (AGMT MBC-10 trial) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT160.

Abstract OT3-07-01: Ixazomib in combination with carboplatin in pretreated women with advanced triple negative breast cancer, an ongoing phase I/II trial (AGMT MBC-10 trial)

Abstract Background:Triple-negative breast cancer (TNBC) comprises a heterogeneous group of diseases which are generally associated with poor prognosis. Recently, the PARP inhibitor olaparib was approved as first targeted treatment beyond antiVEGF therapy for the BRCA1/2 mutated subgroup of TNBC. However, cytotoxic agents still remain the mainstay of treatment for this breast cancer subtype. Ixazomib is a selective and reversible inhibitor of the proteasome, which has been mainly investigated as treatment of multiple myeloma. In a preclinical cell line model for TNBC the first-generation proteasome inhibitor bortezomib showed synergistic efficacy with cisplatin. Clinical data are available for carboplatin plus bortezomib in metastatic ovarian and lung cancers showing remarkable antitumor activity (47% and 38% response rate, respectively) and good tolerability. In solid tumors cytotoxic effect of proteasome inhibitors is thought to be mediated through different mechanisms: (1) Inhibition of the Fanconi Anemia and BRCA1 DNA repair mechanism (2) Inhibition of p53 degradation (3) Inhibition of NF-kappa B signaling cascade. Based on this evidence, the phase I/II MBC-10 trial will evaluate the toxicity profile and efficacy of the oral second-generation proteasome inhibitor ixazomib in combination with carboplatin in patients with advanced TNBC. Trial Design: Patients with metastatic TNBC pretreated with at least one prior line of chemotherapy for advanced disease with a confirmed disease progression and measurable disease are eligible for this study.Patients will receive ixazomib in combination with carboplatin on days 1, 8, and 15 in a 28-day cycle. The phase I part of this study uses an alternate dose escalation accelerated titration design. After establishing the maximum tolerated dose (MTD), accrual continues to evaluate the efficacy and safety of the combination (phase II, including 41 evaluable patients). All patients will continue on study drugs until disease progression, unacceptable toxicity or discontinuation for any other reason. Primary endpoint of the phase II is overall response rate, secondary endpoints include safety profile, progression-free survival and quality of life. The MBC-10 trial is accompanied by a broad biomarker program investigating predictive biomarkers for treatment response and potential resistance mechanisms to the investigational drug combination. This trial is open for patient enrollment since November 2016 in six Austrian cancer centers. Accrual is planned to be completed within two years. ClinicalTrials.gov Identifier: NCT02993094 Citation Format: Rinnerthaler G, Gampenrieder SP, Petzer A, Burgstaller S, Voskova D, Rossmann D, Balic M, Egle D, Rumpold H, Singer CF, Petru E, Melchardt T, Ulmer H, Mlineritsch B, Greil R. Ixazomib in combination with carboplatin in pretreated women with advanced triple negative breast cancer, an ongoing phase I/II trial (AGMT MBC-10 trial) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT3-07-01.

Emodin: One Main Ingredient of Shufeng Jiedu Capsule Reverses Chemoresistance of Lung Cancer Cells Through Inhibition of EMT

Background: Chemoresistance has become a an important worldwide problem to cancer treatment. Understanding the mechanism of drug resistance is the key to solve this problem and improve the survival of the patient. Doxorubicin and its analogues are widely used as antitumor drugs but many doxorubicin resistant cases have been identified in recent years. Doxorubicin (Dox) resistance is a very serious phenomenon in lung cancer treatment. As we could show previously, Shufeng Jiedu Capsule (SFJDC) can effectively reverse H69AR cells resistance to Dox, thus, the present study was designed to explore the mechanism underlying the effects of the main ingredient Emodin on chemosensitivity of H69AR cells to Dox. Methods: First, the growth inhibition rate of lung cancer cells and normal bronchial epithelial cells (BECs) was determined by MTT. Then, the resistance-induced epithelial-mesenchymal transition (EMT) of H69AR cells was examined by western blot and the effect of Emodin on Twist, Snail or Slug was assayed by Real-time PCR and Western blot. The activation of NF-kappa B was assayed by Western blot. Proliferation, apoptosis, migration and invasion of H69AR cells induced by Twist, Snail and Slug were also assayed by flow cytometry and transwell chamber. Results: The results showed that after administration of Dox (10µM) with different concentrations of Emodin, the cells exhibited a dose-dependent inhibition action to H69AR cells at 48 hours. H69AR induced the expression of Twist, Snail, and Slug when compared with Dox-sensitive H69 cells. The expression of Twist, Snail, and Slug can be effectively inhibited by combination of Dox and Emodin. The reversal of resistance was associated with the inhibition of NF-kappa B. Twist, Snail and Slug promoted proliferation, migration and invasion and inhibited apoptosis. Conclusion: Our data suggest that Emodin can effectively reverse the resistance of H69AR to Dox, an effect paralleled by inhibition of EMT, cell proliferation, apoptosis, migration and invasion.

Inhibition of NF-kappa B pathway leads to deregulation of epithelial–mesenchymal transition and neural invasion in pancreatic cancer

NF-κB has an essential role in the initiation and progression of pancreatic cancer and specifically mediates the induction of epithelial–mesenchymal transition and invasiveness. In this study, we demonstrate the importance of activated NF-κB signaling in EMT induction, lymphovascular metastasis, and neural invasion. Modulation of NF-κB activity was accomplished through the specific NF-κB inhibitor (BAY 11-7085), triptolide, and Minnelide treatment, as well as overexpression of IKBα repressor and IKK activator plasmids. In the classical lymphovascular metastatic cascade, inhibition of NF-κB decreased the expression of several EMT transcription factors (SNAI1, SNAI2, and ZEB1) and mesenchymal markers (VIM and CDH2) and decreased in vitro invasion, which was rescued by IKK activation. This was further demonstrated in vivo via BAY 11-7085 treatment in a orthotopic model of pancreatic cancer. In vivo NF-κB inhibition decreased tumor volume; decreased tumor EMT gene expression, while restoring cell–cell junctions; and decreasing overall metastasis. Furthermore, we demonstrate the importance of active NF-κB signaling in neural invasion. Triptolide treatment inhibits Nerve Growth Factor (NGF) mediated, neural-tumor co-culture in vitro invasion, and dorsal root ganglia (DRG) neural outgrowth through a disruption in tumor-neural cross talk. In vivo, Minnelide treatment decreased neurotrophin expression, nerve density, and sciatic nerve invasion. Taken together, this study demonstrates the importance of NF-κB signaling in the progression of pancreatic cancer through the modulation of EMT induction, lymphovascular invasion, and neural invasion.

Effect of recombinant human erythropoietin expressions of apoptosis genes in rats following traumatic brain injury

Purpose: To explore the effect of recombinant human erythropoietin (r-HuEPO) on apoptosis in rats after traumatic brain injury.Methods: A total of 48 traumatic brain-injured Sprague Dawley (SD) rats were obtained by improved Feeney’s traumatic brain injury model, and were randomly divided into four groups: normal salinetreated rats (control) and rats treated with r-HuEPO at doses of 1000 U/kg, 3000 U/kg and 5000 U/kg. Brain tissues were collected on the 7th day after trauma surgery. Apoptotic cells, and NF-kappa B (NFĸB)-, c-myc-, and Fas/Fasl-positive cells were identified in brain tissues by immunohistochemical assay.Results: After treatment with r-HuEPO (3000 and 5000 U/kg), expression of NF-κB and Fas/Fasl were significantly decreased (p < 0.05) compared to control rats, especially at the 5000 U/kg dose (p < 0.01). However, for c-myc, no significant difference was observed between r-HuEPO treatment and control groups (p > 0.05). Compared to the 1000 U/kg r-HuEPO group, Fas/Fasl expression levels were significantly lower in the 3000 and 5000 U/kg r-HuEPO groups (p < 0.05). Additionally, expression of NF-κB and Fasl in the 5000 U/kg r-HuEPO group was significantly lower than that in the 3000 U/kg r- HuEPO group (p < 0.05). Moreover, the number of apoptotic cells in the r-HuEPO group (5000 U/kg) was significantly lower than in the control group (p < 0.05).Conclusion: Thus, r-HuEPO may be beneficial for treating traumatic brain injury via inhibition of NFkappa B and Fas/Fasl expressions.Keywords: Recombinant human erythropoietin, NF-kappa B, Traumatic brain injury, Apoptosis, Neuronal damage, Fas/Fasl expression

MiR-155-5p inhibition promotes the transition of bone marrow mesenchymal stem cells to gastric cancer tissue derived MSC-like cells via NF-κB p65 activation.

Gastric cancer tissue-derived MSC-like cells (GC-MSC) share similar characteristics to bone marrow MSC (BM-MSC); however, the phenotypical and functional differences and the molecular mechanism of transition between the two cell types remain unclear. Compared to BM-MSC, GC-MSC exhibited the classic phenotype of reactive stroma cells, a stronger gastric cancer promoting capacity and lower expression of miR-155-5p. Inhibition of miR-155-5p by transfecting miRNA inhibitor induced a phenotypical and functional transition of BM-MSC into GC-MSC-like cells, and the reverse experiment deprived GC-MSC of tumor-promoting phenotype and function. NF-kappa B p65 (NF-κB p65) and inhibitor of NF-kappa B kinase subunit epsilon (IKBKE/IKKε) were identified as targets of miR-155-5p and important for miRNA inhibitor activating NF-κB p65 in the transition. Inactivation of NF-κB by pyrrolidine dithiocarbamic acid (PDTC) significantly blocked the effect of miR-155-5p inhibitor on BM-MSC. IKBKE, NF-κB p65 and phospho-NF-κB p65 proteins were highly enriched in MSC-like cells of gastric cancer tissues, and the latter two were correlated with the pathological progression of gastric cancer. In GC-MSC, the expression of miR-155-5p was downregulated and NF-κB p65 protein was increased and activated. NF-κB inactivation by PDTC or knockdown of its downstream cytokines reversed the phenotype and function of GC-MSC. Taken together, our findings revealed that miR-155-5p downregulation induces BM-MSC to acquire a GC-MSC-like phenotype and function depending on NF-κB p65 activation, which suggests a novel mechanism underlying the cancer associated MSC remodeling in the tumor microenvironment and offers an effective target and approach for gastric cancer therapy.

Indomethacin induced gene regulation in the rat hippocampus.

BackgroundNon-steroidal anti-inflammatory drugs such as indomethacin are widely used to treat inflammatory diseases and manage pain, fever and inflammation in several conditions, including neuropsychiatric disorders. Although they predominantly function by inhibiting cyclooxygenase (COX) activity, important COX-independent actions also occur. These actions could be responsible for the adverse side effects associated with chronic and/or high dose usage of this popular drug class.ResultsWe examined gene regulation in the hippocampus after peripheral administration of indomethacin by employing a microarray approach. Secondary confirmation and the brain expression pattern of regulated genes was examined by in situ hybridization and immunohistochemistry. Transglutaminase 2, serum glucocorticoid inducible kinase, Inhibitor of NF-kappa B and vascular endothelial growth factor were among genes that were prominently upregulated, while G-protein coupled receptor 56 and neuropeptide Y were among genes that were downregulated by indomethacin. Co-localization studies using blood vessel markers revealed that transglutaminase 2 was induced specifically in brain vasculature.ConclusionsThe data demonstrate that COX-inhibitors can differentially regulate gene transcription in multiple, functionally distinctly cell types in the brain. The results provide additional insight into the molecular actions of COX-inhibitors and indicate that their effects on vasculature could influence cerebral blood flow mechanisms.

CgIκB3, the third novel inhibitor of NF-kappa B (IκB) protein, is involved in the immune defense of the Pacific oyster, Crassostrea gigas.

Inhibitor of NF-κB (IκB), the important regulator of NF-κB/Rel signaling pathway, plays the crucial role in immune response of both vertebrates and invertebrates. Here, a novel homologue of IκB was cloned from Crassostrea gigas, and designated as CgIκB3. The complete CgIκB3 cDNA was 1282 bp in length, including a 942 bp open reading frame (ORF), a 51 bp 5' UTR and a 289 bp 3' UTR. The ORF encodes a putative protein of 313 amino acids with a predicted molecular weight of approximately 34.7 kDa. Sequence analysis reveals that CgIκB3 contains a conserved degradation motif but with only five ankyrin repeats. Neither a PEST domain nor a C-terminal casein kinase II phosphorylation site was identified through either alignment or bioinformatic prediction. Phylogenetic analysis suggested that CgIκB3 shares common ancestor with CgIκB1 rather CgIκB2, and theoretically it may originate from one duplication event prior to divergence of CgIκB1 and CgIκB2. Tissue expression analyses demonstrated that CgIκB3 mRNA is the most abundant in gills and heart. The expression following PAMP infection showed that CgIκB3 was significantly up-regulated in a similar pattern when challenged with LPS, HKLM or HKVA, respectively. Moreover, similar to CgIκB1 and CgIκB2, CgIκB3 can also inhibit Rel dependent NF-κB activation in HEK293 cells in a dose-dependent manner. In summary, these findings suggest that CgIκB3 can be as the functional inhibitor of NF-κB/Rel and involved in the host defense of C. gigas. The discovery of the third IκB emphasizes the complexity and importance of the regulation on NF-κB activation.

Inhibition of NF-kappa B signaling restores responsiveness of castrate-resistant prostate cancer cells to anti-androgen treatment by decreasing androgen receptor-variant expression.

Androgen receptor splicing variants (ARVs) which lack the ligand-binding domain (LBD) are associated with the development of castration-resistant prostate cancer (CRPC), including resistance to the new generation of high affinity anti-androgens. However, the mechanism by which ARVs expression is regulated is not fully understood. In this study, we show that activation of classical NF-κB signaling increases the expression of ARVs in prostate cancer (PCa) cells and converts androgen sensitive PCa cells to become androgen insensitive; while, downregulation of NF-κB signaling inhibits ARVs expression and restores responsiveness of CRPC to anti-androgen therapy. In addition, we demonstrated that combination of anti-androgen with NF-κB targeted therapy inhibits efficiently tumor growth of human CRPC xenografts. These results indicate that induction ARVs by activated NF-κB signaling in PCa cells is a critical mechanism by which the PCa progresses to CRPC. This has important implications since it can prolong the survival of CRPC patients by restoring the tumors to once again respond to conventional androgen-deprivation therapy (ADT).

Histamine induces upregulated expression of histamine receptors and increases release of inflammatory mediators from microglia.

Histamine is a potent mediator of inflammation and a regulator of innate and adaptive immune responses. However, the influence of histamine on microglia, the resident immune cells in the brain, remains uninvestigated. In the present study, we found that microglia can constitutively express all four histamine receptors (H1R, H2R, H3R, and H4R), and the expression of H1R and H4R can be selectively upregulated in primary cultured microglia in a dose-dependent manner by histamine. Histamine can also dose-dependently stimulate microglia activation and subsequently production of proinflammatory factors tumor necrosis factor (TNF)-alpha and interleukin-6 (IL-6). The antagonists of H1R and H4R but not H2R and H3R reduced histamine-induced TNF-alpha and IL-6 production, MAPK and PI3K/AKT pathway activation, and mitochondrial membrane potential loss in microglia, suggesting that the actions of histamine are via H1R and H4R. On the other hand, inhibitors of JNK, p38, or PI3K suppressed histamine-induced TNF-alpha and IL-6 release from microglia. Histamine also activated NF-kappa B and ammonium pyrrolidinedithiocarbamate, an inhibitor of NF-kappa B, and reduced histamine-induced TNF-alpha and IL-6 release. In summary, the present study identifies the expression of histamine receptors on microglia. We also demonstrate that histamine induced TNF-alpha and IL-6 release from activated microglia via H1R and H4R-MAPK and PI3K/AKT-NF-kappa B signaling pathway, which will deepen the understanding of microglia-mediated neuroinflammatory symptoms of chronic neurodegenerative disease.

High density lipoproteins improve insulin sensitivity in high-fat diet-fed mice by suppressing hepatic inflammation

Obesity-induced liver inflammation can drive insulin resistance. HDL has anti-inflammatory properties, so we hypothesized that low levels of HDL would perpetuate inflammatory responses in the liver and that HDL treatment would suppress liver inflammation and insulin resistance. The aim of this study was to investigate the effects of lipid-free apoAI on hepatic inflammation and insulin resistance in mice. We also investigated apoAI as a component of reconstituted HDLs (rHDLs) in hepatocytes to confirm results we observed in vivo. To test our hypothesis, C57BL/6 mice were fed a high-fat diet (HFD) for 16 weeks and administered either saline or lipid-free apoAI. Injections of lipid-free apoAI twice a week for 2 or 4 weeks with lipid-free apoAI resulted in: i) improved insulin sensitivity associated with decreased systemic and hepatic inflammation; ii) suppression of hepatic mRNA expression for key transcriptional regulators of lipogenic gene expression; and iii) suppression of nuclear factor κB (NF-κB) activation. Human hepatoma HuH-7 cells exposed to rHDLs showed suppressed TNFα-induced NF-κB activation, correlating with decreased NF-κB target gene expression. We conclude that apoAI suppresses liver inflammation in HFD mice and improves insulin resistance via a mechanism that involves a downregulation of NF-κB activation.

Abstract 1300: The potential role of NF-κB activation in the immortalization of nasopharyngeal epithelial cells.

Abstract Nasopharyngeal carcinoma (NPC) is a common cancer among Southern Chinese. Many molecular and cellular events, particularly those occurring at the early stage of the NPC development, have not been well defined. Immortalization is a pre-requisite property of cancer cells and is regarded as an early event in human carcinogenesis. Defining and understanding these molecular and cellular events could provide us important insights to the early events involved in the carcinogenesis process. Our lab has established multiple immortalized nasopharyngeal epithelial (NP) cell lines using telomerase alone or in combination with other genetic elements including Bmi-1, cyclin D1 and knocking down p16. We observed that NF-Kappa B are commonly activated in immortalized cells compared to primary cells. Immunofluorescence staining and western blotting assay revealed activation of canonical NF-kappa B pathway in these immortalized nasopharyngeal epithelial cell lines with nuclear translocation of NF-kappa B p65 subunit. Interestingly, EGFR is also commonly expressed in our immortalized nasopharyngeal epithelial cells and may contribute to the constitutive activation of NF-kappa B in immortalized nasopharyngeal epithelial cells. Noteworthy, Epstein - Barr Virus (EBV) infection is closely associated with NPC development. There are studies reporting that activation of NF-kappa B and overexpression of EGFR are derived by EBV encoded LMP1 in NPC (Miller et al. Oncogene. 16, 1998). Our data also show LMP1 could induce NF-kappa B activation and EGFR overexpression in NPC cell line. Thus, the activation of NF-kappa B and overexpression of EGFR may be an important component of EBV infection in epithelial cells and contributing to the development of NPC. We further showed that activation of NF-kappa B signaling appears to be essential for the survival of immortalized nasopharyngeal epithelial cells as inhibition of NF-kappa B activity by a specific inhibitor (Nemo binding domain peptide), could selectively inhibit growth of the immortalized nasopharyngeal epithelial cells but not in primary nasopharyngeal epithelial cells. To study the functional interaction of NF-kappa B activation with EGFR, we have examined if EGFR overexpression may support immortalization of nasopharyngeal epithelial cells. We observed that functional EGFR could facilitate cell growth and protect cells from apoptosis. We are now exploring if activation of NF-kappa B and EGFR overexpression may be functionally related during immortalization of nasopharyngeal epithelial cells appears. Citation Format: Dandan Zhu, Yim Ling Yip, Wen Deng, Chi Man Tsang, Lai Man Cheung, Sai Wah Tsao. The potential role of NF-κB activation in the immortalization of nasopharyngeal epithelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1300. doi:10.1158/1538-7445.AM2013-1300 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Immunomodulatory drugs lenalidomide and pomalidomide inhibit multiple myeloma-induced osteoclast formation and the RANKL/OPG ratio in the myeloma microenvironment targeting the expression of adhesion molecules

Multiple myeloma (MM)-induced osteoclast (OC) formation is mainly due to an imbalance of the receptor activator NF-κB ligand (RANKL)-osteoprotegerin (OPG) ratio in favor of RANKL in the bone microenvironment and to the CCL3 production by MM cells. The purpose of the study was to investigate the effect of the immunomodulatory drugs on RANKL/OPG ratio, the production of pro-osteoclastogenic cytokines, and MM-induced OC formation. We found that invivo concentrations of both lenalidomide (LEN) and pomalidomide (POM) significantly blunted RANKL upregulation normalizing the RANKL/OPG ratio in human osteoprogenitor cells (PreOBs) when co-cultured with MM cells and also inhibited CCL3 production by MM cells. A reduction in CD49d expression, a molecule critically involved in RANKL upregulation in the MM microenvironment, accompanied this effect. Consistently, the pro-osteoclastogenic property of MM cells co-cultured with PreOBs was reduced by both LEN and POM. We further investigated the effect of these drugs on the transcriptional profile of both MM cells and PreOBs by microarray analysis, which showed that adhesion molecules, such as ITGA8 and ICAM2, are significantly downregulated in MM cells. Our data suggest that LEN and POM inhibit MM-induced OC formation through normalization of the RANKL/OPG ratio targeting the expression of adhesion molecules by MM cells.

Intracellular NAD+ Depletion Enhances Bortezomib-Induced Myeloma Cytotoxicity

AbstractAbstract 330 Background:Our previous study demonstrated that inhibition of nicotinamide phosphoribosyltransferase (Nampt) acts by severely depleting intracellular NAD+ content and thus eliciting mitochondrial dysfunction and autophagic MM cell death. The proteasome inhibitor Bortezomib induces anti-MM activity by affecting a variety of signaling pathways. However, as with other agents, dose-limiting toxicities and the development of resistance limit its long-term utility. Here, we demonstrate that combining Nampt inhibitor and bortezomb induces synergistic anti-MM cell death both in vitro using MM cell lines or patient CD138+ MM cells and in vivo in a human plasmacytoma xenograft mouse model. Material and Methods:We utilized MM.1S, MM.1R, RPMI-8226, and U266 human MM cell lines, as well as purified tumor cells from patients relapsing after prior therapies. Cell viability and apoptosis assays were performed using Annexin V/PI staining. Intracellular NAD+ level and proteasome activity were quantified after 12, 24, and 48h exposure to single/combination drugs by specific assays. In vitro angiogenesis was assessed by Matrigel capillary-like tube structure formation assay. Immunoblot analysis was performed using antibodies to caspase-8, caspase-9, caspase-3, PARP, Bcl-2, and tubulin. CB-17 SCID male mice (n = 28; 7 mice/EA group) were subcutaneously inoculated with 5.0 × 106 MM.1S cells in 100 microliters of serum free RPMI-1640 medium. When tumors were measurable (3 weeks after MM cell injection), mice were treated for three weeks with vehicle alone, FK866 (30mg/kg 4 days weekly), Bortezomib (0.5 mg/kg twice weekly), or FK866 (30 mg/kg) plus Bortezomib (0.5 mg/kg). Statistical significance of differences observed in FK866, Bortezomib or combination-treated mice was determined using a Student t test. Isobologram analysis was performed using “CalcuSyn” software program. A combination index < 1.0 indicates synergism. Results/Discussion:Combining FK866 and Bortezomib induces synergistic anti-MM activity in vitro against MM cell lines (P<0.005, CI < 1) or patient CD138-positive MM cells (P< 0.004). FK866 plus Bortezomib-induced synergistic effect is associated with: 1)activation of caspase-8, caspase-9, caspase-3, and PARP; 2) improved intracellular NAD+ dissipation; 3) suppression of chymotrypsin-like, caspase-like, and trypsin-like proteolytic activities; 4) inhibition of NF-kappa B signaling; and 5) inhibition of angiogenesis. Importantly, the ectopic overexpression of Nampt rescues this observed synergistic effect; conversely, Nampt knockdown by RNAi significantly enhances the anti-MM effect of bortezomib. In the murine xenograft MM model, low dose combination FK866 (30 mg/kg) and Bortezomib (0.5 mg/kg) is well tolerated, significantly inhibits tumor growth (P < 0.001), and prolongs host survival (2–2.5 months in mice receiving combined drugs, P = 0.001). These findings demonstrate that intracellular NAD+ levels represent a major determinant in the ability of bortezomib to induce apoptosis of MM cells, providing the rationale for clinical protocols evaluating FK866 together with Bortezomib to improve patient outcome in MM. Disclosures:Munshi:Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy.

Collagen XVII (BP180) modulates keratinocyte expression of the proinflammatory chemokine, IL‐8

Collagen XVII (COL17), a transmembrane protein expressed in epidermal keratinocytes (EK), is targeted by pathogenic autoantibodies in bullous pemphigoid. Treatment of EK with anti-COL17 autoantibodies triggers the production of proinflammatory cytokines. In this study, we test the hypothesis that COL17 is involved in the regulation of the EK proinflammatory response, using IL-8 expression as the primary readout. The absence of COL17 in EK derived from a junctional epidermolysis bullosa patient or shRNA-mediated knockdown of COL17 in normal EK resulted in a dysregulation of IL-8 responses under various conditions. The COL17-deficient cells showed an abnormally high IL-8 response after treatment with lipopolysaccharide (LPS), ultraviolet-B radiation or tumor necrosis factor, but exhibited a blunted IL-8 response to phorbol 12-myristate 13-acetate exposure. Induction of COL17 expression in COL17-negative EK led to a normalization of the LPS-induced proinflammatory response. Although α6β4 integrin was found to be up-regulated in COL17-deficient EK, siRNA-mediated knockdown of the α6 and β4 subunits revealed that COL17's effects on the LPS IL-8 response are not dependent on this integrin. In LPS-treated cells, inhibition of NF-kappa B activity in COL17-negative EK resulted in a normalization of their IL-8 response, and expression of an NF-kappa B-driven reporter was shown to be higher in COL17-deficient, compared with normal EK. These findings support the hypothesis that COL17 plays an important regulatory role in the EK proinflammatory response, acting largely via NF-kappa B. Future investigations will focus on further defining the molecular basis of this novel control network.

Dietary Agents in Cancer Chemoprevention and Treatment

Cancer chemoprevention using natural or synthetic compounds to prevent or suppress the development of cancer, is an area of active investigation. Many compounds belonging to diverse chemical classes have been identified as potential chemopreventive agents, including vitamins and minerals, naturally occurring phytochemicals, and synthetic compounds. Understanding the molecular mechanisms of cancer chemoprevention is not only important for the safe application of these compounds in populations of patients at high risk for cancer, but also allows for further development of novel treatment regimens for cancer patients. This special issue contains original research as well as review articles that are intended to stimulate the continuing efforts to understand the use of dietary agents in cancer chemoprevention and treatment. The lead article by S. N. Saldanha and T. O. Tollefsbol provides a comprehensive review of dietary agents that have shown strong chemopreventive and therapeutic properties in vitro. They also discuss the design and modification of these bioactive compounds for pre-clinical and clinical applications. Dietary intake of foods rich in antioxidant compounds has been suggested to be cancer protective. However, randomized clinical trials and epidemiologic studies on the association between intake of foods rich in antioxidants and cancer incidence have yielded mixed results. M. Y. Wei and E. L. Giovannuci discuss the epidemiologic considerations of lycopene as a chemopreventive agent, including measurement of lycopene, its major source in the diet, and the assessment of prostate cancer incidence and progression, with particular emphasis on the effect of PSA screening on this association. K. Zhou and J. J. Raffoul discuss the composition and cancer-protective effects of major phenolic antioxidants in grape skin and grape seed extracts. M. A. Parasramka and S. V. Gupta provide original research demonstrating the anticancer properties of garcinol alone, or combined with curcumin, on pancreatic cancer cells. Garcinol, a polyisoprentylated benzophenone extracted from the rind of the fruit Garcinia indica, a plant found in tropical regions, has antioxidant and anti-inflammatory properties and its role as anticancer agent is thoroughly discussed in the review from N. Saadat and S. V. Gupta. Two manuscripts discussing the effect of dietary agents on DNA repair capacity are also part of this special issue. In a manuscript by J. J. Raffoul et al., the potential for targeting the DNA base excision repair enzyme APE1/Ref-1 using dietary agents such as soy isoflavones, resveratrol, curcumin, ascorbate, and alpha-tocopherol is discussed. The potential for these natural compounds to be combined with chemotherapy or radiotherapy for the more effective treatment of cancer are also reviewed. A proposed mechanism of action is discussed and an attempt is made to delineate which of the two activities of APE1/Ref-1 (DNA repair versus redox activation of cellular transcription factors) is responsible for the observed effects. The second manuscript by R. Rosati et al. reviews the role for dietary folate in the prevention of colorectal cancer. Data are presented which demonstrate that inhibition of DNA repair is protective in the development of preneoplastic colon lesions, both when folate is depleted and when it is not. This manuscript is a comprehensive review of the literature and provides a critical analysis of the experimental designs used in folate and colorectal cancer research. Two additional manuscripts detailing the ability of dietary agents to sensitize cancer cells to chemotherapy and radiotherapy are included in this special issue. An original research article by S. Duangmano et al. demonstrates that curcurbitacin B, a plant phytochemical, inhibited breast cancer cell proliferation in a dose-dependent manner and caused radiosensitization of human breast cancer cells via G2/M cell cycle arrest. Furthermore, an original research article by K. Sahin et al. demonstrate that genistein, a soy isoflavone, sensitizes cervical cancer cells to cisplatin via inhibition of NF-kappa B and Akt/mTOR cell signaling pathways. This special issue concludes with a report of a clinical study demonstrating the prevention of anthracycline-induced cardiac toxicity through supplementation with selenium in a group of pediatric cancer patients. Research efforts aimed at understanding the role of dietary agents and phytochemicals in cancer prevention and treatment are likely to yield high-impact results that have the potential for immediate clinical applications. Furthermore, combination of phytochemicals and nutritional agents with therapies for advanced cancers, including radiotherapy and chemotherapy, would benefit from a complementary and safe approach using dietary agents to mitigate the adverse effects of these therapies on normal tissues while enhancing the therapeutic efficacy. Elucidation of the mechanisms of interaction between dietary agents and conventional cancer treatments will have a major impact on understanding the molecular mechanisms of cancer chemoprevention and will ultimately result in clinical use of dietary agents as an adjunct to standard cancer treatment. Julian J. Raffoul Omer Kucuk Fazlul H. Sarkar Gilda G. Hillman

In vivo and in vitro inhibitory effects of a traditional Chinese formulation on LPS-stimulated leukocyte–endothelial cell adhesion and VCAM-1 gene expression

Aim of the studyA traditional Chinese formulation Huang-Lian-Jie-Du-Tang (HLJDT) exerts anti-inflammatory effects. The present study aimed to investigate the effect of HLJDT on the LPS-stimulated leukocyte–endothelial cell adhesion and VCAM-1 gene expression both in vivo and in vitro. Materials and methodsHLJDT was extracted from rhizoma coptidis, radix scutellariae, cortex phellodendri and fructus gardeniae in a weight rario of 1:1:1:1. In vivo leukocyte–endothelial cell adhesion was observed in rat lung after LPS stimulation (5mg/kg, i.p.) with or without HLJDT (350 or 700mg/kg, i.g.) pretreatment. The protein expression of vascular cell adhesion molecule 1 (VCAM-1) was analyzed by immunohistochemical method. In vitro leukocyte–endothelial cell adhesion was performed by examining the adhesion of THP-1cells to LPS-stimulated human vascular endothelial cells with or without HLJDT pretreatment. The VCAM-1 expression at the RNA and protein levels was investigated by RT-PCR and western blot analysis, respectively. The activation of NF-κB was examined by the nuclear translocation of NF-κB by immunocytochemical method. ResultsIn vivo, HLJDT dose-dependently reduced the number of leukocytes adhered to endothelium and VCAM-1 protein expression in lung venules of LPS-challenged rats. In vitro, HLJDT dose-dependently decreased the number of THP-1cells adhered to LPS-stimulated endothelial cells and the expression of VCAM-1 both at the RNA and protein levels. The LPS-induced nuclear translocation of NF-kappa B in endothelial cells was also dose-dependently inhibited by HLJDT. ConclusionsThe present study demonstrated an additional mechanism underlying the anti-inflmmatory effect of HLJDT by inhibiting the leukocyte–endothelial cell adhesion and VCAM-1 gene expression. The inhibition of NF-kappa B activation by HLJDT might suggest a profound anti-inflammatory consequences.

Attenuation of liver pro-inflammatory responses by Zingiber officinale via inhibition of NF-kappa B activation in high-fat diet-fed rats.

The aim of this study was to investigate whether treatment with a ginger (Zingiber officinale) extract of high-fat diet (HFD)-fed rats suppresses Nuclear factor-kappa B (NF-κB)-driven hepatic inflammation and to subsequently explore the molecular mechanisms in vitro. Adult male Sprague-Dawley rats were treated with an ethanolic extract of Zingiber officinale (400 mg/kg) along with a HFD for 6 weeks. Hepatic cytokine mRNA levels, cytokine protein levels and NF-κB activation were measured by real-time PCR, Western blot and an NF-κB nuclear translocation assay, respectively. In vitro, cell culture studies were carried out in human hepatocyte (HuH-7) cells by treatment with Zingiber officinale (100 μg/mL) for 24 hr prior to interleukin-1β (IL-1β, 8 ng/mL)-induced inflammation. We showed that Zingiber officinale treatment decreased cytokine gene TNFα and IL-6 expression in HFD-fed rats, which was associated with suppression of NF-κB activation. In vitro, Zingiber officinale treatment decreased NF-κB-target inflammatory gene expression of IL-6, IL-8 and serum amyloid A1 (SAA1), while it suppressed NF-κB activity, IκBα degradation and IκB kinase (IKK) activity. In conclusion, Zingiber officinale suppressed markers of hepatic inflammation in HFD-fed rats, as demonstrated by decreased hepatic cytokine gene expression and decreased NF-κB activation. The study demonstrates that the anti-inflammatory effect of Zingiber officinale occurs at least in part through the NF-κB signalling pathway.

P124 Alpha-type 1 polarized dendritic cells loaded with apoptotic allogeneic breast cancer cells can induce potent cytotoxic T lymphocytes against breast cancer

oxidase activity. At the same time, nuclear expression of NF-kappa;B proteins (p50 and p65) and cellular expression of protein products of the NF-kappa;B-dependent genes (c-myc, bcl-xl, inos, cox-2) were decreased but expression of the NF-kappa;B inhibitor protein (IuBa) and p53 protein was enhanced in the same tumors. PPh ability to strengthen antitumor effect of the synthetic inhibitors of PA synthesis – polyhexamethyleneguanidine (PMG) and a-difluoromethylornithine (DFMO) – was tested. GTE was shown to be able to sum up its antitumoral effect with PMG one and, being combined with DFMO, to prolong lifetime of tumor-bearing animals. Conclusion: GTE and GTEW, can essentially retard experimental tumor growth. Their molecular pathways include inhibition of PA synthesis and interconversion and also inhibition of NF-kappa;B activation and expression of NF-kappa;B-dependent oncogenes. PPh can strengthen antitumoral effect of PMG and have antioxidant and antitoxic properties. Therefore, GTE and GTEW are perspective for cancer prevention in the cancer risk groups and for treatment of cancer patients. Work is support by STCU, grant #4894. Disclosure of Interest: None Declared