Upstream Signaling Cascades Research Articles

Core signaling pathways in ovarian cancer stem cell revealed by integrative analysis of multi-marker genomics data

Tumor recurrence occurs in more than 70% of ovarian cancer patients, and the majority eventually becomes refractory to treatments. Ovarian Cancer Stem Cells (OCSCs) are believed to be responsible for the tumor relapse and drug resistance. Therefore, eliminating ovarian CSCs is important to improve the prognosis of ovarian cancer patients. However, there is a lack of effective drugs to eliminate OCSCs because the core signaling pathways regulating OCSCs remain unclear. Also it is often hard for biologists to identify a few testable targets and infer driver signaling pathways regulating CSCs from a large number of differentially expression genes in an unbiased manner. In this study, we propose a straightforward and integrative analysis to identify potential core signaling pathways of OCSCs by integrating transcriptome data of OCSCs isolated based on two distinctive markers, ALDH and side population, with regulatory network (Transcription Factor (TF) and Target Interactome) and signaling pathways. We first identify the common activated TFs in two OCSC populations integrating the gene expression and TF-target Interactome; and then uncover up-stream signaling cascades regulating the activated TFs. In specific, 22 activated TFs are identified. Through literature search validation, 15 of them have been reported in association with cancer stem cells. Additionally, 10 TFs are found in the KEGG signaling pathways, and their up-stream signaling cascades are extracted, which also provide potential treatment targets. Moreover, 40 FDA approved drugs are identified to target on the up-stream signaling cascades, and 15 of them have been reported in literatures in cancer stem cell treatment. In conclusion, the proposed approach can uncover the activated up-stream signaling, activated TFs and up-regulated target genes that constitute the potential core signaling pathways of ovarian CSC. Also drugs and drug combinations targeting on the core signaling pathways might be able to eliminate OCSCs. The proposed approach can also be applied for identifying potential activated signaling pathways of other types of cancers.

Syk-Mediated Suppression of Inflammatory Responses by Cordyceps bassiana.

The fruit body of artificially cultivated Cordyceps bassiana has been reported to exhibit anti-inflammatory and anticancer activities. Although it has been suggested that the fruit body has neutraceutic and pharmaceutic biomaterial potential, the exact anti-inflammatory molecular mechanism has not been fully elucidated. In this study, we demonstrated the immunopharmacologic activity of Cordyceps bassiana under in vitro conditions and investigated its anti-inflammatory mechanism. Water extract (Cm-WE) of the fruit body of artificially cultivated Cordyceps bassiana without polysaccharide fractions reduced the expression of the proinflammatory genes cyclooxygenase (COX)-2, interleukin (IL)-12, and inducible nitric oxide synthase (iNOS) and promoted the expression of the anti-inflammatory gene IL-10 in lipopolysaccharide (LPS)-treated RAW264.7 cells. In addition, this fraction suppressed proliferation and interferon (IFN)-[Formula: see text] production in splenic T lymphocytes. Cm-WE blocked the activation of nuclear factor (NF)-[Formula: see text]B and activator protein (AP)-1 and their upstream inflammatory signaling cascades, including Syk, MEK, and JNK. Using kinase assays, Syk was identified as the target enzyme most strongly inhibited by Cm-WE. These results strongly suggest that Cm-WE suppresses inflammatory responses by inhibiting Syk kinase activity, with potential implications for novel neutraceutic and pharmaceutic biomaterials.

TRPC Channel Downstream Signaling Cascades.

The family of TRP channel is comprised of a large group of cation-permeable channels, displaying as signaling integrators for sensing extracellular stimulus and initiating intracellular signaling cascades. This chapter offers a brief review of the signaling molecules related to TRPC channels, the first identified mammalian TRP family. Besides the signaling molecules involved in TRPC activation, I will focus on their upstream and downstream signaling cascades and the molecules involved in their intracellular trafficking.

Direct Myosin Activation by Omecamtiv Mecarbil for Heart Failure with Reduced Ejection Fraction.

Myosin is the indispensable molecular motor that utilizes chemical energy to produce force for contraction within the cardiac myocyte. Myosin activity is gated by intracellular calcium levels which are regulated by multiple upstream signaling cascades that can be altered for clinical utility using inotropic medications. In contrast to clinically available cardiac inotropes, omecamtiv mecarbil is a novel direct myosin activator developed to augment left ventricular systolic function without the undesirable secondary effects of altered calcium homeostasis. Its identification and synthesis followed high-throughput screening of a reconstituted sarcomere, deliberate optimization, exquisite biochemical evaluation, and subsequently promising effects in animal models were demonstrated. Physiologically, it prolonged the duration of left ventricular systole in animal models, healthy adults, and patients with heart failure with reduced ejection fraction (HFrEF) without changing the velocity of pressure development, as assessed in animal models. It has been formulated for both intravenous and oral administration, and in both acute and chronic settings produced similar alterations in the duration of systole associated with beneficial increases in cardiac output, improvements in left ventricular volumes, and reductions in heart rate and often of natriuretic peptides. Small, asymptomatic increases in troponin were also observed in the absence of clinically evident ischemia. Clinically, the question remains as to whether the possible harm of this minimal troponin release is outweighed by the potential benefits of reduced neurohormonal activation, increased stroke volume and cardiac output, and improved ventricular remodeling in patients treated with omecamtiv mecarbil. The resolution of this question is being addressed by a phase III outcomes trial of this potential novel therapy for heart failure.

Galangin Activates the ERK/AKT-Driven Nrf2 Signaling Pathway to Increase the Level of Reduced Glutathione in Human Keratinocytes.

Previously, we demonstrated that galangin (3,5,7-trihydroxyflavone) protects human keratinocytes against ultraviolet B (UVB)-induced oxidative damage. In this study, we investigated the effect of galangin on induction of antioxidant enzymes involved in synthesis of reduced glutathione (GSH), and investigated the associated upstream signaling cascades. By activating nuclear factor-erythroid 2-related factor (Nrf2), galangin treatment significantly increased expression of glutamate-cysteine ligase catalytic subunit (GCLC) and glutathione synthetase (GSS). This activation of Nrf2 depended on extracellular signal-regulated kinases (ERKs) and protein kinase B (AKT) signaling. Inhibition of GSH in galangin-treated cells attenuated the protective effect of galangin against the deleterious effects of UVB. Our results reveal that galangin protects human keratinocytes by activating ERK/AKT-Nrf2, leading to elevated expression of GSH-synthesizing enzymes.

Pyrrole-Derivative of Chalcone, (E)-3-Phenyl-1-(2-Pyrrolyl)-2-Propenone, Inhibits Inflammatory Responses via Inhibition of Src, Syk, and TAK1 Kinase Activities.

(E)-3-Phenyl-1-(2-pyrrolyl)-2-propenone (PPP) is a pyrrole derivative of chalcone, in which the B-ring of chalcone linked to β-carbon is replaced by pyrrole group. While pyrrole has been studied for possible Src inhibition activity, chalcone, especially the substituents on the B-ring, has shown pharmaceutical, anti-inflammatory, and anti-oxidant properties via inhibition of NF-κB activity. Our study is aimed to investigate whether this novel synthetic compound retains or enhances the pharmaceutically beneficial activities from the both structures. For this purpose, inflammatory responses of lipopolysaccharide (LPS)-treated RAW264.7 cells were analyzed. Nitric oxide (NO) production, inducible NO synthase (iNOS) and tumor necrosis factor-α (TNF-α) mRNA expression, and the intracellular inflammatory signaling cascade were measured. Interestingly, PPP strongly inhibited NO release in a dose-dependent manner. To further investigate this anti-inflammatory activity, we identified molecular pathways by immunoblot analyses of nuclear fractions and whole cell lysates prepared from LPS-stimulated RAW264.7 cells with or without PPP pretreatment. The nuclear levels of p50, c-Jun, and c-Fos were significantly inhibited when cells were exposed to PPP. Moreover, according to the luciferase reporter gene assay after cotransfection with either TRIF or MyD88 in HEK293 cells, NF-κB-mediated luciferase activity dose-dependently diminished. Additionally, it was confirmed that PPP dampens the upstream signaling cascade of NF-κB and AP-1 activation. Thus, PPP inhibited Syk, Src, and TAK1 activities induced by LPS or induced by overexpression of these genes. Therefore, our results suggest that PPP displays anti-inflammatory activity via inhibition of Syk, Src, and TAK1 activity, which may be developed as a novel anti-inflammatory drug.

Mechanistic insights of O-GlcNAcylation that promote progression of cholangiocarcinoma cells via nuclear translocation of NF-κB

O-GlcNAcylation, an O-linked protein glycosylation with a single molecule of N-acetylglucosamine (GlcNAc), is reversibly controlled by O-GlcNAc transferase (OGT) and N-acetyl D-glucosaminidase (OGA). Aberrant O-GlcNAcylation contributes an important role in initiation and progression of many human cancers. Elevation of O-GlcNAcylation in tumor tissues and poor prognosis of cholangiocarcinoma (CCA) patients have been reported. In this study, the role of O-GlcNAcylation in promoting tumor progression was further investigated in CCA cell lines. Suppression of O-GlcNAcylation using small interfering RNAs of OGT (siOGT) significantly reduced cell migration and invasion of CCA cells whereas siOGA treated cells exhibited opposite effects. Manipulating levels of O-GlcNAcylation did affect the nuclear translocation of NF-κB and Akt-phosphorylation together with expression of matrix-metalloproteinases (MMPs). O-GlcNAcylation and nuclear translocation of NF-κB, the upstream signaling cascade of MMP activation were shown to be important for MMP activation. Immunoprecipitation revealed the elevation of O-GlcNAc-modified NF-κB with increased cellular O-GlcNAcylation. Involvement of O-GlcNAcylation in MMP-mediated migration and invasion of CCA cells was shown to be via O-GlcNAcylation and nuclear translocation of NF-κB. This information indicates the significance of O-GlcNAcylation in controlling the metastatic ability of CCA cells, hence, O-GlcNAcylation and its products may be new targets for treatment of metastatic CCA.

Stomagenesis versus myogenesis: Parallels in intrinsic and extrinsic regulation of transcription factor mediated specialized cell-type differentiation in plants and animals.

Although the last common unicellular ancestor of plants and animals diverged several billion years ago, and while having developed unique developmental programs that facilitate differentiation and proliferation specific to plant and animal systems, there still exists a high degree of conservation in the logic regulating these developmental processes within these two seemingly diverse kingdoms. Stomatal differentiation in plants involves a series of orchestrated cell division events mediated by a family of closely related bHLH transcription factors (TFs) to create a pair of mature guard cells. These TFs are in turn regulated by a number of upstream signaling components that ultimately function to achieve lineage specific differentiation and organized tissue patterning on the plant epidermis. The logic involved in the specification of the myogenic differentiation program in animals is intriguingly similar to stomatal differentiation in plants: Closely-related myogenic bHLHs, known as MRFs (Myogenic Regulatory Factors) provide lineage specificity essential for cell-fate determination. These MRFs, similar to the bHLHs in plants, are regulated by several upstream signaling cascades that succinctly regulate each differentiation step, leading to the production of mature muscle fibers. This review aims at providing a perspective on the emerging parallels in the logic employed by key bHLH transcription factors and their upstream signaling components that function to precisely regulate key cell-state transition events in the stomatal as well as myogenic cell lineages.

δ and γ tocotrienols suppress human hepatocellular carcinoma cell proliferation via regulation of Ras-Raf-MEK-ERK pathway-associated upstream signaling.

Tocotrienol (T3) has recently gained increasing interest due to its anti-cancer effect. Here, we investigated the modulating effect of δ and γ T3 on the Ras-Raf-MEK-ERK oncogenic upstream signaling pathway in human hepatocellular carcinoma HepG2 cells. The results indicated that T3 regulated the upstream signaling cascades of this pathway.

The regulation of autophagy during exercise in skeletal muscle.

The merits of exercise on muscle health and well-being are numerous and well documented. However, the mechanisms underlying the robust adaptations induced by exercise, particularly on mitochondria, are less clear and much sought after. Recently, an evolutionary conserved cellular recycling mechanism known as autophagy has been implicated in the adaptations to acute and chronic exercise. A basal level of autophagy is constantly ongoing in cells and tissues, ensuring cellular clearance and energy homeostasis. This pathway can be further induced, as a survival mechanism, by cellular perturbations, such as energetic imbalance and oxidative stress. During exercise, a biphasic autophagy response is mobilized, leading to both an acute induction and a long-term potentiation of the process. Posttranslational modifications arising from upstream signaling cascades induce an acute autophagic response during a single bout of exercise by mobilizing core autophagy machinery. A transcriptional program involving the regulators Forkhead box O, transcription factor EB, p53, and peroxisome proliferator coactivator-1α is also induced to fuel sustained increases in autophagic capacity. Autophagy has also been documented to mediate chronic exercise-induced metabolic benefits, and animal models in which autophagy is perturbed do not adapt to exercise to the same extent. In this review, we discuss recent developments in the field of autophagy and exercise. We specifically highlight the molecular mechanisms activated during acute exercise that lead to a prolonged adaptive response.

A novel quinazolinone chalcone derivative induces mitochondrial dependent apoptosis and inhibits PI3K/Akt/mTOR signaling pathway in human colon cancer HCT-116 cells

We have synthesized a novel quinazolinone chalcone derivative (QC) and first time reported its in-vitro and in-vivo anticancer potential. It inhibited the cell proliferation of different cancer cell lines like PC-3, Panc-1, Mia-Paca-2, A549, MCF-7 and HCT-116. It induces apoptosis as measured by several biological endpoints such as apoptotic body formation, evident by Hoechst and scanning electron microscopy, enhanced annexinV-FITC binding of the cells, increased sub-G0 cell fraction, loss of mitochondrial membrane potential (Δψm), reduction of Bcl-2/Bax ratio, activation of caspase-9, caspase-3 and PARP-1 (poly-ADP Ribose polymerase) cleavage in HCT-116 cells. In spite of apoptosis, QC significantly hammers the downstream and upstream signaling cascade of PI3K/Akt/mTOR pathway and cell cycle regulator Skp-2, p21 and p27. Interestingly, QC induces the S and G2/M phase of HCT-116 cells at experimental doses. QC inhibits the tumor growth of Ehrlich ascites carcinoma (EAC), Ehrlich tumor (ET, solid) and sarcoma-180(solid) mice models. Furthermore, it was found to be non-toxic as no animal mortality (0/7) occurred during experimental doses. The present study provides an insight of anticancer potential of QC, which may be useful in managing and treating cancer.

Kinase-KCC2 coupling: Cl- rheostasis, disease susceptibility, therapeutic target.

The intracellular concentration of Cl(-) ([Cl(-)]i) in neurons is a highly regulated variable that is established and modulated by the finely tuned activity of the KCC2 cotransporter. Despite the importance of KCC2 for neurophysiology and its role in multiple neuropsychiatric diseases, our knowledge of the transporter's regulatory mechanisms is incomplete. Recent studies suggest that the phosphorylation state of KCC2 at specific residues in its cytoplasmic COOH terminus, such as Ser940 and Thr906/Thr1007, encodes discrete levels of transporter activity that elicit graded changes in neuronal Cl(-) extrusion to modulate the strength of synaptic inhibition via Cl(-)-permeable GABAA receptors. In this review, we propose that the functional and physical coupling of KCC2 to Cl(-)-sensitive kinase(s), such as the WNK1-SPAK kinase complex, constitutes a molecular "rheostat" that regulates [Cl(-)]i and thereby influences the functional plasticity of GABA. The rapid reversibility of (de)phosphorylation facilitates regulatory precision, and multisite phosphorylation allows for the control of KCC2 activity by different inputs via distinct or partially overlapping upstream signaling cascades that may become more or less important depending on the physiological context. While this adaptation mechanism is highly suited to maintaining homeostasis, its adjustable set points may render it vulnerable to perturbation and dysregulation. Finally, we suggest that pharmacological modulation of this kinase-KCC2 rheostat might be a particularly efficacious strategy to enhance Cl(-) extrusion and therapeutically restore GABA inhibition.

Abstract A10: The myc post-translational landscape: How novel gain-of-function mutants are revealing new stability and functional regulatory systems

Abstract The c-MYC (MYC) oncogene plays an important role in tumorigenesis and is implicated in >50% of all human cancers. Deregulation of MYC can occur through abnormally high expression levels, but also through oncogenic lesions in upstream signaling cascades. The study of these signaling pathways have provided an alternative approach for the development of MYC-targeted therapeutics. For example, the study of post-translational modifications (PTMs) of MYC, such as P-T58 and the T58A gain-of-function mutant, identified FBXW7 as a tumor suppressor and the deubiquitinating enzyme USP28 as a therapeutic target. We considered that MYC is highly modified post-translationally and that unknown mechanistic pathways may be modifying residues, in addition to T58, in order to control MYC stability and/or function. These undiscovered pathways may therefore provide additional opportunities for the development of MYC-targeted therapeutics. These considerations led to recent work in the Penn lab that uncovered clusters of negatively regulating residues of MYC function. These residues include S71/S81, a cluster of residues referred to as MYC-4 (T343, S344, S347 and S348) and a cluster of 6 lysine residues (6K) at the C-terminal end of MYC (K298, K317, K323, K326, K341 and K355). These negatively regulating residues were characterized using alanine (S71/S81 and 340 cluster) and arginine (C-terminal lysines) substitution mutants in our established transformation assays. The S71/S81A and MYC-4A mutants scored with having gain-of-function activity in comparison to wild-type MYC in multiple transformation assays including growth in soft agar and the disruption of regular acini formation using a normal, immortalized MCF10A cell line. In addition, these mutants were shown to regulate additional genes compared to wild-type MYC using genome-wide mRNA expression analysis of MCF10A acini, suggesting that these MYC proteins have gained additional transcriptional targets. Additionally, substitution of the C-terminal lysine residues with arginine (6KR) also revealed gain-of-function activity. 6KR expressing MCF10A and SH-EP cells had increased anchorage-independent growth compared to cells expressing wild-type MYC and was also more potent in promoting xenograft tumor growth of Rat1A and SH-EP cells. Interestingly, all three mutants do not have extended half-lives as seen with T58A, suggesting that functional activity and not stability is contributing to these transformative phenotypes. The above mutants reveal that each of S71/S81, MYC-4 and C-terminal 6K residues are critically important for the negative regulation of MYC-induced transformation. To further explore these regions of MYC, we used mass spectrometry to identify post-translational modifications that occurred on MYC in growing cells. These data confirm phosphorylation events on S71/81 as well as at MYC-4A. Strikingly, three modifications were directly observed on three of the six lysine residues; acetylation of lysine 323, ubiquitylation of lysine 355 and SUMOylation of lysine 326. The importance of these modifications and the roles that these modifications have in regulating MYC activity are currently under investigation using our established transformation assays. I now aim to understand the contribution of single or multiple modifications within the indicated clusters and how these modifications modulate MYC activity. Citation Format: Corey Lourenco, Amanda Wasylishen, Michelle Chan-Seng-Yue, Christina Bros, Dharmendra Dingar, William Tu, Manpreet Kalkat, Pak-Kei Chan, Peter Mullen, Brian Raught, Paul Boutros, Linda Penn. The myc post-translational landscape: How novel gain-of-function mutants are revealing new stability and functional regulatory systems. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A10.

Abstract 4384: TET inhibits phosphorylation of eIF4E-binding protein, in prostate cancer cells

Abstract Introduction and Objectives: All eukaryotic cellular mRNAs are blocked at their 5-prime ends with the 7-methyl-guanosine cap structure. EIF4E is a eukaryotic translation initiation factor involved in directing ribosomes to the cap structure of mRNAs. The EIF4E polypeptide is the rate-limiting component of the eukaryotic translation apparatus and is involved in the mRNA-ribosome binding step of eukaryotic protein synthesis. Tetrandrine (TET) is a bis-benzylisoquinoline alkaloid, isolated from the root of Stefenia tatrandra. Previous studies have shown that TET has anti-inflammatory, immunologic and antiallergenic effects. We have shown that TET targets AR signaling in PCa cells and induces expression of p21. However the molecular mechanism by which TET exerts its effects are not completely understood. In the present study we investigated the effects of TET in prostate cancer cells in culture (PC-3 and LNCaP cells). Methods: LNCaP, C-4 and PC3 cells were grown in supplemented RPMI media and DMEM/F12 Media respectively, and maintained at 37C in a 5%CO2 incubator (as described previously). Protein levels were measured by Western Blot and by immuno-fluorescence assays. In vitro translation system from HeLa cell lysates was used to measure protein synthesis in vitro. Protein Synthesis in vivo was measured by a reduction in the rate of firefly luciferase production following introduction of luciferase mRNA into cells by nucleofection. mRNA expression measured in RTPCR assays. Promoter activities were measured by measuring promoter driven luciferase expression. Results: TET inhibited protein synthesis in dose-dependent way with IC50 value of 20 μM as measured by a reduction in the rate of firefly luciferase production following introduction of luciferase mRNA into cells by nucleofection. The inhibition of protein synthesis was 88% at 40 μM. Interestingly TET did not inhibit protein synthesis in in vitro translation system from HeLa cell lysates. This suggested that inhibition by TET occurs in signaling pathways regulating protein synthesis that are operative in intact cells but not cell-free lysates. It is well known that un-phosphorylated 4E-BP1 binds with high affinity to cap-binding protein eIF4E, thus preventing recruitment of 48S translation initiation complex to capped mRNA and inhibiting protein synthesis. By western blotting, we demonstrated that tetrandrine inhibited phosphorylation of translational repressor 4E-BP1 at Ser65, the substrate of multiple upstream signaling cascades. Conclusions: Our results suggest that TET is a novel agent to target EIF4E and that Tet inhibits EIF4. Current research is aimed at determining the involvement of other upstream signaling pathways that might explain inhibition of 4E-BP1 phosphorylation following TET treatment.Financial support : (VA-1BX001258; NCI RO1-161880; Carroll W. Feist Endowed Chair Funds (Koul H), Funds from, FWCC, the Dean School SOM and Chancellor- LSUHSC-Shreveport. Citation Format: Sweaty Koul, Qin Dong, Sergey Slepenkov, Hari K. Koul. TET inhibits phosphorylation of eIF4E-binding protein, in prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4384. doi:10.1158/1538-7445.AM2015-4384

Intracellular Sensing and Transport of Amino Acids and the Downstream Effects on mTORC1 Activation: Implications for Muscle Protein Synthesis

It is widely established that the rate of muscle protein synthesis is elevated with amino acid availability. However, when there is a lack in amino acid availability homeostatic processes are implemented in order to prioritize the energy demands necessitating amino acid scarcity. To this respect, it is understood that amino acid availability is a rate-limiting factor underlying the mediation of muscle protein synthesis (MPS). The mammalian target of rapamycin complex 1 (mTORC1) is a master regulatory complex that plays a significant role in meticulously coordinating the myriad of signaling cascades in order to up-regulate MPS activity through increased protein translation initiation. The presence of adequate amino acids such as leucine is evidenced to be necessary for the activation of mTORC1. Moreover, amino acids have been known as subsequent mediators that trigger mTORC1 activation independent of the traditional upstream signaling cascades associated with growth factors. It has recently been observed that the activation of mTORC1 is implemented at the surface of the lysosome membrane. As a result, considerable advances have occurred which demonstrate various protein components which relay on amino acids to relay signals to mTORC1. This review will focalize upon describing the present understanding of the multiple signaling components that mediate signals in conditions of adequate amino acid levels to mTORC1. Furthermore, this review will also present recent advancements behind amino acid sensing mechanisms through the discussion of upstream components of mTORC1, amino acid transporters, as well as proposed underlying mechanisms of intracellular signaling relative to amino acid sensing.

(E)-3-(3,4-dihydroxy-2-methoxyphenyl)-1-(2,4-dihydroxyphenyl)prop-2-en-1-one, a novel licochalcone B derivative compound, suppresses lipopolysaccharide-stimulated inflammatory reactions in RAW264.7 cells and endotoxin shock in mice

Activated macrophages mediate inflammation, as they release nitric oxide and pro-inflammatory cytokines in various inflammatory diseases. Suppressing macrophage activation may alleviate inflammatory processes. Here, we report that (E)-3-(3,4-dihydroxy-2-methoxyphenyl)-1-(2,4-dihydroxyphenyl)prop-2-en-1-one (DDP), a novel licochalcone B derivative compound, inhibits inflammatory reactions in macrophages and protects mice from endotoxin shock. In vitro experiments showed that DDP suppressed the generation of nitric oxide and pro-inflammatory cytokines by suppressing the activation of nuclear factor-κB and activator protein-1 and simultaneously inhibited its upstream inflammatory signaling cascades in lipopolysaccharide in RAW264.7 cells. In an animal model, DDP protected BALB/c mice from lipopolysaccharide-induced endotoxin shock, possibly through inhibition of the production of inflammatory cytokines. DDP inhibited the production of inflammatory mediators and may be a potential target for treatment of various inflammatory diseases.

Abstract 2454: Ampk and hdac pathways and regulation rassf1c gene expression

Abstract Introduction: We have previously shown that RASSF1C promotes cell proliferation, migration and attenuates apoptosis in cancer cells. We have also shown that RASSF1C is significantly up-regulated in breast and lung cancer tissues compared to normal tissues. Together these demonstrate that RASSF1C, is not a tumor suppressor like RASSF1A, but instead appears to function as an oncoprotein. Currently nothing is known about the upstream signaling cascades involved in regulating RASSF1C gene expression and thus we performed transcriptome PCR array study to identify chemicals inhibitors that modulate(s) RASSF1C expression. Method: A transcriptome PCR array was obtained from Qiagen. It consisted of cDNA from breast cancer cell line MCF7 cells treated with 90 different chemical inhibitors that regulate various signaling pathways. The array was screened with RASSF1C gene specific primers. Data analysis was performed by importing the Ct values obtained into data analysis software. Results: The PCR array screen identified several chemical reagents that appear to up-regulate and several that seem to down-regulate RASSF1C expression by ≥ 1.5 fold. The two most notable reagents are Dorsopmorhin (AMPK inhibitor) which up-regulates RASSF1C expression by 2.8 fold and Trichostatin A (HDAC inhibitor) which down regulates RASSF1C expression by 2 fold. We are in process of confirming the effect of these inhibitors in a panel of breast and lung cancer cells. Conclusion: The findings are novel and suggest that inhibition of AMPK pathway induces RASSF1C expression while inhibition of HDAC pathway suppresses RASSF1C gene expression. We should note that Trichostatin A has been shown to activate AMPK, inhibits HDAC, arrests cell growth, and induces apoptosis in human cancers. Our findings provide a potential mechanism for regulating RASSF1C gene expression through the modulation of AMPK and HDAC pathways. Citation Format: Yousef G. Amaar, Mark E. Reeves, Matthew Firek. Ampk and hdac pathways and regulation rassf1c gene expression. [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 2454. doi:10.1158/1538-7445.AM2014-2454

Protein kinase C-associated kinase regulates NF-κB activation through inducing IKK activation.

Activation of the transcription factor NF-κB induced by extracellular stimuli requires IKKα and IKKβ kinase activity. How IKKα and IKKβ are activated by various upstream signaling molecules is not fully understood. We previously showed that protein kinase C-associated kinase (PKK, also known as DIK/RIP4), which belongs to the receptor-interacting protein (RIP) kinase family, mediates the Bcell activating factor of the TNF family (BAFF)-induced NF-κB activation in diffuse large B cell lymphoma (DLBCL) cell lines. Here we have investigated the mechanism underlying NF-κB activation regulated by PKK. Our results suggest that PKK can activate both the classical and the alternative NF-κB activation pathways. PKK associates with IKKα and IKKβ in mammalian cells and induces activation of both IKKα and IKKβ via phosphorylation of their serine residues 176/180 and 177/181, respectively. Unlike other members of the RIP family that activate NF-κB through a kinase-independent pathway, PKK appears to activate IKK and NF-κB mainly in a kinase-dependent manner. Suppression of PKK expression by RNA interference inhibits phosphorylation of IKKα and IKKβ as well as activation of NF-κB in human cancer cell lines. Thus, PKK regulates NF-κB activation by modulating activation of IKKα and IKKβ in mammalian cells. We propose that PKK may provide a critical link between IKK activation and various upstream signaling cascades, and may represent a potential target for inhibiting abnormal NF-κB activation in human cancers.

Inhibition of FoxO1 nuclear exclusion prevents metastasis of glioblastoma.

Glioblastoma is the most aggressive malignant primary brain tumor in humans, with extremely poor patient survival. Although previous studies have demonstrated that expression of matrix metalloproteinase-9 (MMP9) in glioblastoma promotes cancer metastasis, the upstream molecular signaling cascades that control activation of MMP9 remain largely unknown. Here, we used a human glioblastoma line, A-172, to examine molecular signaling to activate MMP9. We found that epidermal growth factor (EGF)-induced activation of epidermal growth factor receptor (EGFR) in A-172 cells activated MMP9, resulting in an increase in cancer invasiveness. A specific inhibitor for EGFR efficiently blocked EGF-induced activation of MMP9 and then cancer invasiveness. Moreover, an inhibitor for phosphatidylinositol 3-kinase (PI-3 K)/protein kinase B (Akt) significantly inhibited the EGF-induced activation of MMP9. Furthermore, nuclear exclusion of a major Akt downstream target, Forkhead box protein O1 (FoxO1), was induced by Akt activation, which could be inhibited by either an EGFR inhibitor or by PI-3 K/Akt inhibitor. An expression of a constitutive nuclear form of FoxO1 significantly inhibited MMP9 activation induced by EGF. Taken together, these findings suggest that EGF/EGFR signaling activates downstream PI-3 K/Akt to induce FoxO1 nuclear exclusion, which activates MMP9 to promote glioblastoma invasiveness. Thus, FoxO1 appears to be a novel therapeutic target for inhibiting metastasis of glioblastoma.

(5-Hydroxy-4-oxo-4H-pyran-2-yl)methyl 6-hydroxynaphthalene-2-carboxylate, a kojic acid derivative, inhibits inflammatory mediator production via the suppression of Syk/Src and NF-κB activation

Numerous derivatives of kojic acid have been synthesised to expand its immunopharmacological uses. Kojic acid is known to have anti-cancer, anti-inflammatory, and anti-melanogenesis effects. We found that (5-hydroxy-4-oxo-4H-pyran-2-yl)methyl 6-hydroxynaphthalene-2-carboxylate (MHNC) strongly suppressed the production of nitric oxide (NO) in an initial screening experiment. In this study, we explored the in vitro and in vivo anti-inflammatory activity of MHNC and its inhibitory mechanisms using lipopolysaccharide (LPS)-treated RAW264.7 cells and HCl/EtOH-treated ICR mice. MHNC dose-dependently diminished the secretion of nitric oxide (NO) and prostaglandin (PG)E2 in LPS-treated RAW264.7 cells. This compound also suppressed the upregulation of mRNA levels for the inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 genes. Additionally, the transcriptional activation of these genes was inhibited by MHNC through the suppression of the nuclear translocation of nuclear factor (NF)-κB subunits (p65 and p50), as determined by a luciferase reporter assay. Interestingly, MHNC treatment was found to suppress a series of upstream signalling cascades consisting of IκBα, AKT, PDK1, Src, and Syk for NF-κB activation. Furthermore, a direct enzyme assay with purified Src and Syk and luciferase assays using Src and Syk overexpression indicated that these enzymes were directly inhibited by MHNC. Finally, MHNC (20mg/kg) prevented inflammatory symptoms of the stomach in mice treated with HCl/EtOH by reducing phospho-IκBα levels. Taken together, our data suggest that MHNC may negatively modulate in vitro and in vivo inflammatory responses via the direct suppression of Syk/Src and NF-κB.