Negative Regulation of Leptin-induced Reactive Oxygen Species (ROS) Formation by Cannabinoid CB1 Receptor Activation in Hypothalamic Neurons

The adipocyte-derived, anorectic hormone leptin was recently shown to owe part of its regulatory effects on appetite-regulating hypothalamic neuropeptides to the elevation of reactive oxygen species (ROS) levels in arcuate nucleus (ARC) neurons. Leptin is also known to exert a negative regulation on hypothalamic endocannabinoid levels and hence on cannabinoid CB₁ receptor activity. Here we investigated the possibility of a negative regulation by CB₁ receptors of leptin-mediated ROS formation in the ARC. Through pharmacological and molecular biology experiments we report data showing that leptin-induced ROS accumulation is 1) blunted by arachidonyl-2′-chloroethylamide (ACEA) in a CB₁-dependent manner in both the mouse hypothalamic cell line mHypoE-N41 and ARC neuron primary cultures, 2) likewise blocked by a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, troglitazone, in a manner inhibited by T0070907, a PPAR-γ antagonist that also inhibited the ACEA effect on leptin, 3) blunted under conditions of increased endocannabinoid tone due to either pharmacological or genetic inhibition of endocannabinoid degradation in mHypoE-N41 and primary ARC neuronal cultures from MAGL^−/− mice, respectively, and 4) associated with reduction of both PPAR-γ and catalase activity, which are reversed by both ACEA and troglitazone. We conclude that CB₁ activation reverses leptin-induced ROS formation and hence possibly some of the ROS-mediated effects of the hormone by preventing PPAR-γ inhibition by leptin, with subsequent increase of catalase activity. This mechanism might underlie in part CB1 orexigenic actions under physiopathological conditions accompanied by elevated hypothalamic endocannabinoid levels.     

Overexpression of transforming growth factor type?III receptor restores TGF-β1 sensitivity in human tongue squamous cell carcinoma cells

The transforming growth factor type III receptor (TβRIII), also known as β-glycan, is a multi-functional sensor that regulates growth, migration and apoptosis in most cancer cells. We hereby investigated the expression of TβRIII in clinical specimens of tongue squamous cell carcinoma (TSCC) and the underlying mechanism that TβRIII inhibits the growth of CAL-27 human oral squamous cells. The TSCC tissues showed a significant decrease in TβRIII protein expression as detected by immunohistochemistry (IHC) and western blot analysis. Transfection of TβRIII-containing plasmid DNA dramatically promoted TGF-β1 (10 ng/ml)-induced decrease in cell viability, apoptosis and cell arrest at the G₀-/G₁-phase. Moreover, transient overexpression of TβRIII enhanced the TGF-β1-induced cyclin-dependent kinase inhibitor 2b (CDKN2b) and p38 protein activity, but did not affect the activities of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase 1/2 (JNK1/2) in CAL-27 cells. These results suggest overexpression of TβRIII receptor restored TGF-β1 sensitivity in CAL-27 cells, which may provide some new insights on exploiting this molecule therapeutically.     

An Undesired Effect of Chemotherapy: GEMCITABINE PROMOTES PANCREATIC CANCER CELL INVASIVENESS THROUGH REACTIVE OXYGEN SPECIES-DEPENDENT,NUCLEAR FACTOR κB- AND HYPOXIA-INDUCIBLE FACTOR 1α-MEDIATED UP-REGULATION OF CXCR4*

Recently, we have shown that CXCL12/CXCR4 signaling plays an important role in gemcitabine resistance of pancreatic cancer (PC) cells. Here, we explored the effect of gemcitabine on this resistance mechanism. Our data demonstrate that gemcitabine induces CXCR4 expression in two PC cell lines (MiaPaCa and Colo357) in a dose- and time-dependent manner. Gemcitabine-induced CXCR4 expression is dependent on reactive oxygen species (ROS) generation because it is abrogated by pretreatment of PC cells with the free radical scavenger N-acetyl-L-cysteine. CXCR4 up-regulation by gemcitabine correlates with time-dependent accumulation of NF-κB and HIF-1α in the nucleus. Enhanced binding of NF-κB and HIF-1α to the CXCR4 promoter is observed in gemcitabine-treated PC cells, whereas their silencing by RNA interference causes suppression of gemcitabine-induced CXCR4 expression. ROS induction upon gemcitabine treatment precedes the nuclear accumulation of NF-κB and HIF-1α, and suppression of ROS diminishes these effects. The effect of ROS on NF-κB and HIF-1α is mediated through activation of ERK1/2 and Akt, and their pharmacological inhibition also suppresses gemcitabine-induced CXCR4 up-regulation. Interestingly, our data demonstrate that nuclear accumulation of NF-κB results from phosphorylation-induced degradation of IκBα, whereas HIF-1α up-regulation is NF-κB-dependent. Lastly, our data demonstrate that gemcitabine-treated PC cells are more motile and exhibit significantly greater invasiveness against a CXCL12 gradient. Together, these findings reinforce the role of CXCL12/CXCR4 signaling in gemcitabine resistance and point toward an unintended and undesired effect of chemotherapy.     

Isovalerylspiramycin I suppresses non-small cell lung carcinoma growth through ROS-mediated inhibition of PI3K/AKT signaling pathway

Novel drugs are required for non-small cell lung cancer (NSCLC) treatment urgently. Repurposing old drugs as new treatments is a practicable approach with time and cost savings. Some studies have shown that carrimycin, a Chinese Food and Drug Administration (CFDA)-approved macrolide antibiotic, possesses potent anti-tumor effects against oral squamous cell carcinoma. However, its detailed component and underlying mechanisms in anti-NSCLC remain unknown. In our study, isovalerylspiramycin I (ISP-I) was isolated from carrimycin and demonstrated a remarkable anti-NSCLC efficacy in vitro and in vivo with a favorable safety profile. It has been proven that in NSCLC cell lines H460 and A549, ISP-I could induce G2/M arrest and apoptosis, which was mainly attributed to ROS accumulation and subsequently PI3K/AKT signaling pathway inhibition. Numerous downstream genes including mTOR and FOXOs were also changed correspondingly. An observation of NAC-induced reverse effect on ISP-I-leading cell death and PI3K/AKT pathway inhibition, emphasized the necessity of ROS signaling in this event. Moreover, we identified ROS accumulation and PI3K/AKT pathway inhibition in tumor xenograft models in vivo as well. Taken together, our study firstly reveals that ISP-I is a novel ROS inducer and may act as a promising candidate with multi-target and low biological toxicity for anti-NSCLC treatment.     

RIP3-Dependent Accumulation of Mitochondrial Superoxide Anions in TNF-α-Induced Necroptosis

Excessive production of reactive oxygen species (ROS) is a key phenomenon in tumor necrosis factor (TNF)-α-induced cell death. However, the role of ROS in necroptosis remains mostly elusive. In this study, we show that TNF-α induces the mitochondrial accumulation of superoxide anions, not H₂O₂, in cancer cells undergoing necroptosis. TNF-α-induced mitochondrial superoxide anions production is strictly RIP3 expression-dependent. Unexpectedly, TNF-α stimulates NADPH oxidase (NOX), not mitochondrial energy metabolism, to activate superoxide production in the RIP3-positive cancer cells. In parallel, mitochondrial superoxide-metabolizing enzymes, such as manganese-superoxide dismutase (SOD2) and peroxiredoxin III, are not involved in the superoxide accumulation. Mitochondrial-targeted superoxide scavengers and a NOX inhibitor eliminate the accumulated superoxide without affecting TNF-α-induced necroptosis. Therefore, our study provides the first evidence that mitochondrial superoxide accumulation is a consequence of necroptosis.     

Gemfibrozil,a Lipid-lowering Drug,Increases Myelin Genes in Human Oligodendrocytes via Peroxisome Proliferator-activated Receptor-β

An increase in CNS remyelination and a decrease in CNS inflammation are important steps to halt the progression of multiple sclerosis. Earlier studies have shown that gemfibrozil, a lipid-lowering drug, has anti-inflammatory properties. The current study identified another novel property of gemfibrozil in stimulating the expression of myelin-specific genes (myelin basic protein, myelin oligodendrocyte glycoprotein, 2′,3′-cyclic-nucleotide 3′-phosphodiesterase, and proteolipid protein (PLP)) in primary human oligodendrocytes, mixed glial cells, and spinal cord organotypic cultures. Although gemfibrozil is a known activator of peroxisome proliferator-activated receptor-α (PPAR-α), we were unable to detect PPAR-α in either gemfibrozil-treated or untreated human oligodendrocytes, and gemfibrozil increased the expression of myelin genes in oligodendrocytes isolated from both wild type and PPAR-α(−/−) mice. On the other hand, gemfibrozil markedly increased the expression of PPAR-β but not PPAR-γ. Consistently, antisense knockdown of PPAR-β, but not PPAR-γ, abrogated the stimulatory effect of gemfibrozil on myelin genes in human oligodendrocytes. Gemfibrozil also did not up-regulate myelin genes in oligodendroglia isolated from PPAR-β(−/−) mice. Chromatin immunoprecipitation analysis showed that gemfibrozil induced the recruitment of PPAR-β to the promoter of PLP and myelin oligodendrocyte glycoprotein genes in human oligodendrocytes. Furthermore, gemfibrozil treatment also led to the recruitment of PPAR-β to the PLP promoter in vivo in the spinal cord of experimental autoimmune encephalomyelitis mice and suppression of experimental autoimmune encephalomyelitis symptoms in PLP-T cell receptor transgenic mice. These results suggest that gemfibrozil stimulates the expression of myelin genes via PPAR-β and that gemfibrozil, a prescribed drug for humans, may find further therapeutic use in demyelinating diseases.     

Lysophosphatidic Acid Acyltransferase Beta Regulates mTOR Signaling

Lysophosphatidic acid acyltransferase (LPAAT-β) is a phosphatidic acid (PA) generating enzyme that plays an essential role in triglyceride synthesis. However, LPAAT-β is now being studied as an important regulator of cell growth and differentiation and as a potential therapeutic target in cancer since PA is necessary for the activity of key proteins such as Raf, PKC-ζ and mTOR. In this report we determine the effect of LPAAT-β silencing with siRNA in pancreatic adenocarcinoma cell lines. We show for the first time that LPAAT-β knockdown inhibits proliferation and anchorage-independent growth of pancreatic cancer cells. This is associated with inhibition of signaling by mTOR as determined by levels of mTORC1- and mTORC2-specific phosphorylation sites on 4E-BP1, S6K and Akt. Since PA regulates the activity of mTOR by modulating its binding to FKBP38, we explored the possibility that LPAAT-β might regulate mTOR by affecting its association with FKBP38. Coimmunoprecipitation studies of FKBP38 with mTOR show increased levels of FKBP38 associated with mTOR when LPAAT-β protein levels are knocked down. Furthermore, depletion of LPAAT-β results in increased Lipin 1 nuclear localization which is associated with increased nuclear eccentricity, a nuclear shape change that is dependent on mTOR, further confirming the ability of LPAAT-β to regulate mTOR function. Our results provide support for the hypothesis that PA generated by LPAAT-β regulates mTOR signaling. We discuss the implications of these findings for using LPAAT-β as a therapeutic target.     

Accumulation of Phosphorylated β-Catenin Enhances ROS-Induced Cell Death in Presenilin-Deficient Cells

Presenilin (PS) is involved in many cellular events under physiological and pathological conditions. Previous reports have revealed that PS deficiency results in hyperproliferation and resistance to apoptotic cell death. In the present study, we investigated the effects of PS on β-catenin and cell mortality during serum deprivation. Under these conditions, PS1/PS2 double-knockout MEFs showed aberrant accumulation of phospho-β-catenin, higher ROS generation, and notable cell death. Inhibition of β-catenin phosphorylation by LiCl reversed ROS generation and cell death in PS deficient cells. In addition, the K19/49R mutant form of β-catenin, which undergoes normal phosphorylation but not ubiquitination, induced cytotoxicity, while the phosphorylation deficient S37A β-catenin mutant failed to induce cytotoxicity. These results indicate that aberrant accumulation of phospho-β-catenin underlies ROS-mediated cell death in the absence of PS. We propose that the regulation of β-catenin is useful for identifying therapeutic targets of hyperproliferative diseases and other degenerative conditions.     

Naa10p and IKKα interaction regulates EMT in oral squamous cell carcinoma via TGF‐β1/Smad pathway

Epithelial‐mesenchymal transition (EMT) has been contributed to increase migration and invasion of cancer cells. However, the correlate of Naa10p and IKKα with EMT in oral squamous cell carcinoma (OSCC) is not yet fully understood. In our present study, we found N‐α‐acetyltransferase 10 protein (Naa10p) and IκB kinase α (IKKα) were abnormally abundant in oral squamous cell carcinoma (OSCC). Bioinformatic results indicate that the expression of Naa10p and IKKα is correlated with TGF‐β1/Smad and EMT‐related molecules. The Transwell migration, invasion, qRT‐PCR and Western blot assay indicated that Naa10p repressed OSCC cell migration, invasion and EMT, whereas IKKα promoted TGF‐β1–mediated OSCC cell migration, invasion and EMT. Mechanistically, Naa10p inhibited IKKα activation of Smad3 through the interaction with IKKα directly in OSCC cells after TGF‐β1 stimulation. Notably, knockdown of Naa10p reversed the IKKα‐induced change in the migration, invasion and EMT‐related molecules in OSCC cells after TGF‐β1 stimulation. These findings suggest that Naa10p interacted with IKKα mediates EMT in OSCC cells through TGF‐β1/Smad, a novel pathway for preventing OSCC.     

Isorhamnetin Inhibits Proliferation and Invasion and Induces Apoptosis through the Modulation of Peroxisome Proliferator-activated Receptor γ Activation Pathway in Gastric Cancer

Gastric cancer (GC) is a lethal malignancy and the second most common cause of cancer-related deaths. Although treatment options such as chemotherapy, radiotherapy, and surgery have led to a decline in the mortality rate due to GC, chemoresistance remains as one of the major causes for poor prognosis and high recurrence rate. In this study, we investigated the potential effects of isorhamnetin (IH), a 3′-O-methylated metabolite of quercetin on the peroxisome proliferator-activated receptor γ (PPAR-γ) signaling cascade using proteomics technology platform, GC cell lines, and xenograft mice model. We observed that IH exerted a strong antiproliferative effect and increased cytotoxicity in combination with chemotherapeutic drugs. IH also inhibited the migratory/invasive properties of GC cells, which could be reversed in the presence of PPAR-γ inhibitor. We found that IH increased PPAR-γ activity and modulated the expression of PPAR-γ regulated genes in GC cells. Also, the increase in PPAR-γ activity was reversed in the presence of PPAR-γ-specific inhibitor and a mutated PPAR-γ dominant negative plasmid, supporting our hypothesis that IH can act as a ligand of PPAR-γ. Using molecular docking analysis, we demonstrate that IH formed interactions with seven polar residues and six nonpolar residues within the ligand-binding pocket of PPAR-γ that are reported to be critical for its activity and could competitively bind to PPAR-γ. IH significantly increased the expression of PPAR-γ in tumor tissues obtained from xenograft model of GC. Overall, our findings clearly indicate that antitumor effects of IH may be mediated through modulation of the PPAR-γ activation pathway in GC.     

Plasma membrane proteoglycans syndecan-2 and syndecan-4 engage with EGFR and RON kinase to sustain carcinoma cell cycle progression

Epidermal growth factor receptor (EGFR) is a causal factor in carcinoma, yet many carcinoma patients are resistant to EGFR inhibitors. Potential insight into this resistance stems from prior work that showed EGFR in normal epithelial cells docks to the extracellular domain of the plasma membrane proteoglycan syndecan-4 (Sdc4) engaged with α3β1 and α6β4 integrins. We now report that this receptor complex is modified by the recruitment of syndecan-2 (Sdc2), the Recepteur d’Origine Nantais (RON) tyrosine kinase, and the cellular signaling mediator Abelson murine leukemia viral oncogene homolog 1 (ABL1) in triple-negative breast carcinoma and head and neck squamous cell carcinoma, where it contributes to EGFR kinase–independent proliferation. Treatment with a peptide mimetic of the EGFR docking site in the extracellular domain of Sdc4 (called SSTN_EGFR) disrupts the entire complex and causes a rapid, global arrest of the cell cycle. Normal epithelial cells do not recruit these additional receptors to the adhesion mechanism and are not arrested by SSTN_EGFR. Although EGFR docking with Sdc4 in the tumor cells is required, cell cycle progression does not depend on EGFR kinase. Instead, progression depends on RON kinase, activated by its incorporation into the complex. RON activates ABL1, which suppresses p38 mitogen-activated protein kinase and prevents a p38-mediated signal that would otherwise arrest the cell cycle. These findings add to the growing list of receptor tyrosine kinases that support tumorigenesis when activated by their association with syndecans at sites of matrix adhesion and identify new potential targets for cancer therapy.     

Cyclosporin A inhibits colon cancer cell growth independently of the calcineurin pathway

Chronic inflammation is a risk factor for the development of colon cancer, providing genotoxic insults, growth and pro-angiogenic factors that can promote tumorigenesis and tumor growth. Immunomodulatory agents can interfere with the inflammation that feeds cancer, but their impact on the transformed cell is poorly understood. The calcium/calcineurin signaling pathway, through activation of NFAT, is essential for effective immune responses, and its inhibitors cyclosporin A (CsA) and FK506 are used in the clinics to suppress immunity. Moreover, the kinases GSK3β and mTOR, modulated by PI-3K/Akt, can inhibit NFAT activity, suggesting a cross-talk between the calcium and growth factor signaling pathways. Both NFAT and mTOR activity have been associated with tumorigenesis. We therefore investigated the impact of calcineurin and PI-3K/mTOR inhibition in growth of human colon carcinoma cells. We show that despite the efficient inhibition of NFAT1 activity, FK506 promotes tumor growth, whereas CsA inhibits it due to a delay in cell cycle progression and induction of necroptosis. We found NFκB activation and mTORC1 activity not to be altered by CsA or FK506. Similarly, changes to mitochondrial homeostasis were equivalent upon treatment with these drugs. We further show that, in our model, NFAT1 activation is not modulated by PI3K/mTOR. We conclude that CsA slows cell cycle progression and induces necroptosis of human carcinoma cell lines in a TGFβ-, NFAT-, NFκB- and PI3K/mTOR-independent fashion. Nevertheless, our data suggest that CsA, in addition to its anti-inflammatory capacity, may target transformed colon and esophagus carcinoma cells without affecting non-transformed cells, promoting beneficial tumoristatic effects.     

Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Phosphorylation by Protein Kinase Cδ (PKCδ) Inhibits Mitochondria Elimination by Lysosomal-like Structures following Ischemia and Reoxygenation-induced Injury

After cardiac ischemia and reperfusion or reoxygenation (I/R), damaged mitochondria propagate tissue injury by promoting cell death. One possible mechanism to protect from I/R-induced injury is the elimination of damaged mitochondria by mitophagy. Here we identify new molecular events that lead to mitophagy using a cell culture model and whole hearts subjected to I/R. We found that I/R induces glyceraldehyde-3-phosphate dehydrogenase (GAPDH) association with mitochondria and promotes direct uptake of damaged mitochondria into multiorganellar lysosomal-like (LL) structures for elimination independently of the macroautophagy pathway. We also found that protein kinase C δ (PKCδ) inhibits GAPDH-driven mitophagy by phosphorylating the mitochondrially associated GAPDH at threonine 246 following I/R. Phosphorylated GAPDH promotes the accumulation of mitochondria at the periphery of LL structures, which coincides with increased mitochondrial permeability. Either inhibition of PKCδ or expression of a phosphorylation-defective GAPDH mutant during I/R promotes a reduction in mitochondrial mass and apoptosis, thus indicating rescued mitophagy. Taken together, we identified a GAPDH/PKCδ signaling switch, which is activated during oxidative stress to regulate the balance between cell survival by mitophagy and cell death due to accumulation of damaged mitochondria.     

Akt-dependent Activation of mTORC1 Complex Involves Phosphorylation of mTOR (Mammalian Target of Rapamycin) by IκB Kinase α (IKKα)

The serine/threonine protein kinase Akt promotes cell survival, growth, and proliferation through phosphorylation of different downstream substrates. A key effector of Akt is the mammalian target of rapamycin (mTOR). Akt is known to stimulate mTORC1 activity through phosphorylation of tuberous sclerosis complex 2 (TSC2) and PRAS40, both negative regulators of mTOR activity. We previously reported that IκB kinase α (IKKα), a component of the kinase complex that leads to NF-κB activation, plays an important role in promoting mTORC1 activity downstream of activated Akt. Here, we demonstrate IKKα-dependent regulation of mTORC1 using multiple PTEN null cancer cell lines and an animal model with deletion of IKKα. Importantly, IKKα is shown to phosphorylate mTOR at serine 1415 in a manner dependent on Akt to promote mTORC1 activity. These results demonstrate that IKKα is an effector of Akt in promoting mTORC1 activity.     

Muscle Wasting in Fasting Requires Activation of NF-κB and Inhibition of AKT/Mechanistic Target of Rapamycin (mTOR) by the Protein Acetylase,GCN5

NF-κB is best known for its pro-inflammatory and anti-apoptotic actions, but in skeletal muscle, NF-κB activation is important for atrophy upon denervation or cancer. Here, we show that also upon fasting, NF-κB becomes activated in muscle and is critical for the subsequent atrophy. Following food deprivation, the expression and acetylation of the p65 of NF-κB on lysine 310 increase markedly in muscles. NF-κB inhibition in mouse muscles by overexpression of the IκBα superrepressor (IκBα-SR) or of p65 mutated at Lys-310 prevented atrophy. Knockdown of GCN5 with shRNA or a dominant-negative GCN5 or overexpression of SIRT1 decreased p65K310 acetylation and muscle wasting upon starvation. In addition to reducing atrogene expression, surprisingly inhibiting NF-κB with IκBα-SR or by GCN5 knockdown in these muscles also enhanced AKT and mechanistic target of rapamycin (mTOR) activities, which also contributed to the reduction in atrophy. These new roles of NF-κB and GCN5 in regulating muscle proteolysis and AKT/mTOR signaling suggest novel approaches to combat muscle wasting.