Mitochondrial reactive oxygen is critical for IL-12/IL-18-induced IFN-γ production by CD4+ T cells and is regulated by Fas/FasL signaling

Mitochondrial activation and the production of mitochondrial reactive oxygen species (mROS) are crucial for CD4⁺ T cell responses and have a role in naïve cell signaling after TCR activation. However, little is known about mROS role in TCR-independent signaling and in recall responses. Here, we found that mROS are required for IL-12 plus IL-18-driven production of IFN-γ, an essential cytokine for inflammatory and autoimmune disease development. Compared to TCR stimulation, which induced similar levels of mROS in naïve and memory-like cells, IL-12/IL-18 showed faster and augmented mROS production in memory-like cells. mROS inhibition significantly downregulated IFN-γ and CD44 expression, suggesting a direct mROS effect on memory-like T cell function. The mechanism that promotes IFN-γ production after IL-12/IL-18 challenge depended on the effect of mROS on optimal activation of downstream signaling pathways, leading to STAT4 and NF-κB activation. To relate our findings to IFN-γ-driven lupus-like disease, we used Fas-deficient memory-like CD4⁺ T cells from lpr mice. Importantly, we found significantly increased IFN-γ and mROS production in lpr compared with parental cells. Treatment of WT cells with FasL significantly reduced mROS production and the activation of signaling events leading to IFN-γ. Moreover, Fas deficiency was associated with increased mitochondrial levels of cytochrome C and caspase-3 compared with WT memory-like cells. mROS inhibition significantly reduced the population of disease-associated lpr CD44^hiCD62L^loCD4⁺ T cells and their IFN-γ production. Overall, these findings uncovered a previously unidentified role of Fas/FasL interaction in regulating mROS production by memory-like T cells. This apoptosis-independent Fas activity might contribute to the accumulation of CD44^hiCD62L^loCD4⁺ T cells that produce increased IFN-γ levels in lpr mice. Overall, our findings pinpoint mROS as central regulators of TCR-independent signaling, and support mROS pharmacological targeting to control aberrant immune responses in autoimmune-like disease.Subject terms: Autoimmunity, Cytokines     

Mitochondrial ROS--radical detoxification, mediated by protein kinase D

The mitochondrial electron transport chain is the major source for the production of oxygen radicals. Mitochondria-generated reactive oxygen species (mROS) have been implicated in decreasing the life span and contributing to age-related diseases (known as the free radical theory of aging). Recently, the serine/threonine kinase protein kinase D1 (PKD1) was identified as a mitochondrial sensor for oxidative stress. mROS-activated PKD regulates a radical-sensing signaling pathway, which relays mROS production to the induction of nuclear genes that mediate cellular detoxification and survival. This PKD regulated signaling pathway is the first known mitochondria located and mitochondrially regulated antioxidant system that protects these organelles and cells from oxidative stress-mediated damage or cell death. The identification of this and further intracellular protective signaling pathways provides an opportunity to manipulate the effects of mROS, and might provide the key to targeting aging effects and age-related diseases that have been linked to mitochondrial dysfunctions.     

Activation of TLR4 signaling promotes gastric cancer progression by inducing mitochondrial ROS production

Chronic infection, such as Helicobacter pylori infection, has been associated with the development of gastric cancer (GC). Pathogen-associated molecular patterns can trigger inflammatory responses via Toll-like receptors (TLRs) in GC. Here we showed that Toll-like receptor 4 (TLR4) was highly expressed in GC cells and was associated with the aggressiveness of GC. The binding of lipopolysaccharide (LPS) to TLR4 on GC cells enhanced proliferation without affecting apoptosis. Higher level of reactive oxygen species (ROS) was induced after activation of TLR4 signaling in GC. Using oxidase inhibitors and antioxidants, we found that mitochondrial ROS (mROS) was major source of TLR4-stimulated ROS generation. This elevated mROS production can be inhibited by diphenylene iodonium (DPI), and the blocking of the mROS production rather than ROS neutralization resulted in cell cycle arrest and the loss of mitochondrial potential, which were plausible reason for decreased cell viability. Furthermore, the increased mROS owing to TLR4 signaling resulted in the activation of Akt phosphorylation and NF-κB p65 nuclear translocation. Altogether, these results reveal a novel pathway linking innate immune signaling to GC cell proliferation, implicate mROS as an important component of cell survival signals and further establish mitochondria as hubs for GC therapies.     

Angiopreventive efficacy of pure flavonolignans from milk thistle extract against prostate cancer: targeting VEGF-VEGFR signaling

The role of neo-angiogenesis in prostate cancer (PCA) growth and metastasis is well established, but the development of effective and non-toxic pharmacological inhibitors of angiogenesis remains an unaccomplished goal. In this regard, targeting aberrant angiogenesis through non-toxic phytochemicals could be an attractive angiopreventive strategy against PCA. The rationale of the present study was to compare the anti-angiogenic potential of four pure diastereoisomeric flavonolignans, namely silybin A, silybin B, isosilybin A and isosilybin B, which we established previously as biologically active constituents in Milk Thistle extract. Results showed that oral feeding of these flavonolignans (50 and 100 mg/kg body weight) effectively inhibit the growth of advanced human PCA DU145 xenografts. Immunohistochemical analyses revealed that these flavonolignans inhibit tumor angiogenesis biomarkers (CD31 and nestin) and signaling molecules regulating angiogenesis (VEGF, VEGFR1, VEGFR2, phospho-Akt and HIF-1α) without adversely affecting the vessel-count in normal tissues (liver, lung, and kidney) of tumor bearing mice. These flavonolignans also inhibited the microvessel sprouting from mouse dorsal aortas ex vivo, and the VEGF-induced cell proliferation, capillary-like tube formation and invasiveness of human umbilical vein endothelial cells (HUVEC) in vitro. Further studies in HUVEC showed that these diastereoisomers target cell cycle, apoptosis and VEGF-induced signaling cascade. Three dimensional growth assay as well as co-culture invasion and in vitro angiogenesis studies (with HUVEC and DU145 cells) suggested the differential effectiveness of the diastereoisomers toward PCA and endothelial cells. Overall, these studies elucidated the comparative anti-angiogenic efficacy of pure flavonolignans from Milk Thistle and suggest their usefulness in PCA angioprevention.     

Visualizing common deletion of mitochondrial DNA-augmented mitochondrial reactive oxygen species generation and apoptosis upon oxidative stress

Common deletion (CD) 4977 bp of mitochondrial DNA (mtDNA) disrupt specifically mitochondrial complex I, IV and V on the electron transport chain (ETC) and is closely associated with wide spectrums of clinical manifestations. To quantitatively investigate how CD-induced ETC defect alters mitochondrial reactive oxygen species (mROS) generation as well as down stream apoptotic signaling, we employed an established array of human CD cytoplasmic hybrids (cybrids) harboring 0%-80% of CD. Pathological effects of CD on the mitochondria were visualized at single cell level by the application of fluorescent probes coupled with conventional and multiphoton imaging microscopy. Intriguingly, we observed CD-augmented mROS generation omitted "threshold effect". CD-augmented mROS generation was associated with depolarized mitochondrial membrane potential (DeltaPsi(m)). Upon oxidative stress, the amount of CD-augmented mROS generation was greatly enhanced to cause pathological apoptotic deterioration including opening of the mitochondrial permeability transition, cytochrome c release, phosphatidylserine externalization and DNA fragmentation. In addition, heterogeneous mitochondrial dysfunctions were found in cybrids containing 80% of CD (D cybrids), i.e., low sensitive-D (LS-D, roughly 80%) and a super sensitive-D (SS-D, 20%). As compared to LS-D, SS-D had higher resting mROS level but slightly hyperpolarized DeltaPsi(m). Upon H2O2 treatment, much faster generation of mROS was observed which induced a faster depolarization of DeltaPsi(m) and later apoptotic deterioration in SS-D. We proposed a dose-dependent, feed-forward and self-accelerating vicious cycle of mROS production might be initiated in CD-induced ETC defect without threshold effect. As CD-augmented mROS generation is obligated to cause an enhanced pathological apoptosis, precise detection of CD-augmented mROS generation and their degree of heterogeneity in single cells may serve as sensitive pathological indexes for early diagnosis, prognosis and treatment of CD-associated diseases.     

Quercetin Inhibits Angiogenesis Mediated Human Prostate Tumor Growth by Targeting VEGFR- 2 Regulated AKT/mTOR/P70S6K Signaling Pathways

Angiogenesis is a crucial step in the growth and metastasis of cancers, since it enables the growing tumor to receive oxygen and nutrients. Cancer prevention using natural products has become an integral part of cancer control. We studied the antiangiogenic activity of quercetin using ex vivo, in vivo and in vitro models. Rat aortic ring assay showed that quercetin at non-toxic concentrations significantly inhibited microvessel sprouting and exhibited a significant inhibition in the proliferation, migration, invasion and tube formation of endothelial cells, which are key events in the process of angiogenesis. Most importantly, quercetin treatment inhibited ex vivo angiogenesis as revealed by chicken egg chorioallantoic membrane assay (CAM) and matrigel plug assay. Western blot analysis showed that quercetin suppressed VEGF induced phosphorylation of VEGF receptor 2 and their downstream protein kinases AKT, mTOR, and ribosomal protein S6 kinase in HUVECs. Quercetin (20 mg/kg/d) significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model, indicating that quercetin inhibited tumorigenesis by targeting angiogenesis. Furthermore, quercetin reduced the cell viability and induced apoptosis in prostate cancer cells, which were correlated with the downregulation of AKT, mTOR and P70S6K expressions. Collectively the findings in the present study suggest that quercetin inhibits tumor growth and angiogenesis by targeting VEGF-R2 regulated AKT/mTOR/P70S6K signaling pathway, and could be used as a potential drug candidate for cancer therapy.     

Dietary Compound Isoliquiritigenin Inhibits Breast Cancer Neoangiogenesis via VEGF/VEGFR-2 Signaling Pathway

Angiogenesis is crucial for cancer initiation, development and metastasis. Identifying natural botanicals targeting angiogenesis has been paid much attention for drug discovery in recent years, with the advantage of increased safety. Isoliquiritigenin (ISL) is a dietary chalcone-type flavonoid with various anti-cancer activities. However, little is known about the anti-angiogenic activity of isoliquiritigenin and its underlying mechanisms. Herein, we found that ISL significantly inhibited the VEGF-induced proliferation of human umbilical vein endothelial cells (HUVECs) at non-toxic concentration. A series of angiogenesis processes including tube formation, invasion and migration abilities of HUVECs were also interrupted by ISL in vitro. Furthermore, ISL suppressed sprout formation from VEGF-treated aortic rings in an ex-vivo model. Molecular mechanisms study demonstrated that ISL could significantly inhibit VEGF expression in breast cancer cells via promoting HIF-1α (Hypoxia inducible factor-1α) proteasome degradation and directly interacted with VEGFR-2 to block its kinase activity. In vivo studies further showed that ISL administration could inhibit breast cancer growth and neoangiogenesis accompanying with suppressed VEGF/VEGFR-2 signaling, elevated apoptosis ratio and little toxicity effects. Molecular docking simulation indicated that ISL could stably form hydrogen bonds and aromatic interactions within the ATP-binding region of VEGFR-2. Taken together, our study shed light on the potential application of ISL as a novel natural inhibitor for cancer angiogenesis via the VEGF/VEGFR-2 pathway. Future studies of ISL for chemoprevention or chemosensitization against breast cancer are thus warranted.     

Honokiol,a small molecular weight natural product,inhibits angiogenesis in vitro and tumor growth in vivo

Natural products comprise a major source of small molecular weight angiogenesis inhibitors. We have used the transformed endothelial cell line SVR as an effective screen of natural product extracts to isolate anti-angiogenesis and anti-tumor compounds. Aqueous extracts of Magnolia grandiflora exhibit potent activity in our SVR proliferation assays. We found that the small molecular weight compound honokiol is the active principle of magnolia extract. Honokiol exhibited potent anti-proliferative activity against SVR cells in vitro. In addition, honokiol demonstrated preferential inhibition of primary human endothelial cells compared with fibroblasts and this inhibition was antagonized by antibodies against TNF alpha-related apoptosis-inducing ligand. In vivo, honokiol was highly effective against angiosarcoma in nude mice. Our preclinical data suggests that honokiol is a systemically available and non-toxic inhibitor of angiogenesis and should be further evaluated as a potential chemotherapeutic agent.     

Proangiogenic activity of beta-carotene is coupled with the activation of endothelial cell chemotaxis

Endothelial cells play an important role in angiogenesis (formation of new vessels from preexisting ones), which is essential for organogenesis, tissue remodeling but also inflammatory response, carcinogenesis in all periods of our life. Beta-carotene (BC) in non-toxic concentrations (up to 3 microM) had no detectable effect on HUVECs (human umbilical vein endothelial cells) proliferation or apoptosis, despite significant changes of the expression patterns of pro- and anti-apoptotic genes. However beta-carotene did not change the tubulogenic activity of HUVEC in the in vitro angiogenesis model, it potently accelerated the bFGF-induced development of microcapillaries, as well as the migration of endothelial cells, in matrigel plug injected subcutaneously to mice. Potent activation of endothelial cell migration in the in vitro model of chemotaxis was also observed. According to the microarray data, genes involved in cell/cell and cell/matrix adhesion, matrix reorganization, activation of chemotaxis, the G-protein regulated intracellular signaling as well as genes involved in the rapid remodeling of protein cytoskeleton were the most affected by BC in HUVEC. We conclude that beta-carotene in the physiological concentration range stimulates early steps of angiogenesis by the activation of cellular migration as well as matrix reorganization and decrease of cell adhesion.     

Bone Morphogenetic Protein functions as a context-dependent angiogenic cue in vertebrates

Bone Morphogenetic Protein (BMP) signaling has been implicated in diverse biological processes. Although how BMP signaling regulates behaviors of endothelial cells during angiogenesis are not fully understood, increasing evidence indicate functions of BMP signaling components are essential in developmental and pathological angiogenesis. Here we review recent advances in delineating the functions of BMP signaling during angiogenesis. In addition, we discuss downstream pathways that transduce BMP signaling in endothelial cells, and factors that modulate BMP signaling response in endothelial cells. Finally, we provide recent insight on how BMP signaling functions as a context dependent angiogenic cue.     

Functional estrogen receptors in the mitochondria of breast cancer cells

Steroid hormones have been reported to indirectly impact mitochondrial functions, attributed to nuclear receptor-induced production of proteins that localize in this cytoplasmic organelle. Here we show high-affinity estrogen receptors in the mitochondria of MCF-7 breast cancer cells and endothelial cells, compatible with classical estrogen receptors ERalpha and ERbeta. We report that in MCF-7, estrogen inhibits UV radiation-induced cytochrome C release, the decrease of the mitochondrial membrane potential, and apoptotic cell death. UV stimulated the formation of mitochondrial reactive oxygen species (mROS), and mROS were essential to inducing mitochondrial events of cell death. mROS mediated the UV activation of c-jun N-terminal kinase (JNK), and protein kinase C (PKC) delta, underlying the subsequent translocation of Bax to the mitochondria where oligomerization was promoted. E2 (estradiol) inhibited all these events, directly acting in mitochondria to inhibit mROS by rapidly up-regulating manganese superoxide dismutase activity. We implicate novel functions of ER in the mitochondria of breast cancer that lead to the survival of the tumor cells.     

The role of cell adhesion pathways in angiogenesis

Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is prevalent both during normal mammalian development and in certain pathological conditions such as tumor growth. It is stimulated and controlled by a complex network of intracellular signaling mechanisms, many of which are initiated by trans-membrane receptors transducing signals received from other cells and from the extracellular environment. Of these, cytokine signaling is recognized as one of the primary drivers of angiogenesis, but it has become increasingly evident that signaling mechanisms generated as a result of cell adhesion interactions are also crucially important. In addition, cell adhesion pathways are also intimately tied to cytokine signaling often making it difficult to dissect out the relative contribution of each to a particular angiogenic step. Many of these same signaling mechanisms are often manipulated by tumors to stimulate aberrant angiogenesis and enhance their blood supply. As a consequence, there is a great deal of interest in trying to understand the full complement of intracellular signaling pathways in angiogenesis as well as their interplay and timing during the process. Ultimately, understanding the complex network of signaling pathways that function during angiogenesis will provide important avenues for future therapeutic development.     

Regulation of angiogenesis via Notch signaling in breast cancer and cancer stem cells

Breast cancer angiogenesis is elicited and regulated by a number of factors including the Notch signaling. Notch receptors and ligands are expressed in breast cancer cells as well as in the stromal compartment and have been implicated in carcinogenesis. Signals exchanged between neighboring cells through the Notch pathway can amplify and consolidate molecular differences, which eventually dictate cell fates. Notch signaling and its crosstalk with many signaling pathways play an important role in breast cancer cell growth, migration, invasion, metastasis and angiogenesis, as well as cancer stem cell (CSC) self-renewal. Therefore, significant attention has been paid in recent years toward the development of clinically useful antagonists of Notch signaling. Better understanding of the structure, function and regulation of Notch intracellular signaling pathways, as well as its complex crosstalk with other oncogenic signals in breast cancer cells will be essential to ensure rational design and application of new combinatory therapeutic strategies. Novel opportunities have emerged from the discovery of Notch crosstalk with inflammatory and angiogenic cytokines and their links to CSCs. Combinatory treatments with drugs designed to prevent Notch oncogenic signal crosstalk may be advantageous over λ secretase inhibitors (GSIs) alone. In this review, we focus on the more recent advancements in our knowledge of aberrant Notch signaling contributing to breast cancer angiogenesis, as well as its crosstalk with other factors contributing to angiogenesis and CSCs.     

The role of the mitochondrion in plant responses to biotic stress

Recent studies suggest that the plant mitochondrion may play a role during biotic stress responses, such as those occurring during incompatible plant–pathogen interactions. There are indications that signal molecules or pathways initiated by such interactions may directly or indirectly target mitochondrial components and that an important consequence of this targeting is an early disruption of mitochondrial homeostasis, resulting in an increased generation of mitochondrial reactive oxygen species (mROS). These mROS may then initiate further mitochondrial dysfunction and further mROS generation in a self-amplifying manner. The mROS, as well as the graded dysfunction of the mitochondrion may act as cellular signals that initiate graded cellular responses ranging from defense gene induction to initiation of programmed cell death. However, these events may be attenuated by the unique components of the plant electron transport chain that act to substitute for dysfunctional components, dampen mROS generation or facilitate in defining the cellular level of ROS and antioxidant defense systems.     

Melatonin protects hepatocytes against bile acid-induced mitochondrial oxidative stress via the AMPK-SIRT3-SOD2 pathway

Abstract

Mitochondrial oxidative damage is hypothesized to contribute to the pathogenesis of chronic cholestatic liver diseases. Melatonin, an indolamine synthesized in the pineal gland, shows a wide range of physiological functions, and is under clinical investigation for expanded applications. Melatonin has demonstrated efficient protective effects against various types of oxidative damage in the liver system. This study investigates the protective effects of melatonin pretreatment on glycochenodeoxycholic acid (GCDCA)-induced hepatotoxicity and elucidates the potential mechanism of melatonin-mediated protection. Melatonin markedly decreased mitochondrial ROS (mROS) production in L02 cells treated with 100 μM GCDCA, and inhibited GCDCA-stimulated cytotoxicity. Notably, melatonin exerted its hepatoprotective effects by upregulating sirtuin 3 (SIRT3) activity and its expression level, thus regulating superoxide dismutase 2 (SOD2) acetylation and inhibiting the production of mROS induced by GCDCA. Moreover, siRNA targeting SIRT3 blocked the melatonin-mediated elevation in mitochondrial function by inhibiting SIRT3/SOD2 signaling. Importantly, melatonin-activated SIRT3 activity was completely abolished by AMP-activated, alpha 1 catalytic subunit (AMPK) siRNA transfection. Similar results were obtained in rat with bile duct ligation or BDL. In summary, our findings indicate that melatonin is a novel hepatoprotective small molecule that functions by elevating SIRT3, stimulating SOD2 activity, and suppressing mitochondrial oxidative stress at least through AMPK, and that SIRT3 may be of therapeutic value in liver cell protection for GCDCA-induced hepatotoxicity.