Fructose-induced peroxynitrite production is mediated by methylglyoxal in vascular smooth muscle cells

Methylglyoxal (MG), a highly reactive molecule, has been implicated in the development of insulin resistance. We investigated whether fructose, a precursor of MG, induced ONOO(-) generation and whether this process was mediated via endogenously increased MG formation. Fructose significantly increased MG generation in vascular smooth muscle cells (VSMCs) in a concentration and time dependent manner. The intracellular production of MG was increased by 153+/-23% or 259+/-28% after cells were treated 6 h with fructose (15 mM or 30 mM), compared with production from untreated cells (p<0.01, n=4 for each group). A significant increase in the production of ONOO(-), NO, and O(2)(*-), was found in the cells treated with fructose (15 mM) or MG (10 microM). Fructose- or MG-induced ONOO(-) generation was significantly inhibited by MG scavengers, including reduced glutathione or N-acetyl-l-cysteine, and by O(2)(*-) or NO inhibitors, such as diphenylene iodonium, superoxide dismutase or N-nitro-l-arginine methyl ester. Moreover, an enhanced iNOS expression was also observed in the cells treated directly with MG which was significantly inhibited when co-application with N-acetyl-l-cysteine. Our results demonstrated that fructose is capable of inducing a significant increase in ONOO(-) production, which is mediated by an enhanced formation of endogenous MG in VSMCs.     

Anoikis evasion in inflammatory breast cancer cells is mediated by Bim-EL sequestration

Inflammatory breast cancer (IBC) is a rare and highly invasive type of breast cancer, and patients diagnosed with IBC often face a very poor prognosis. IBC is characterized by the lack of primary tumor formation and the rapid accumulation of cancerous epithelial cells in the dermal lymphatic vessels. Given that normal epithelial cells require attachment to the extracellular matrix (ECM) for survival, a comprehensive examination of the molecular mechanisms underlying IBC cell survival in the lymphatic vessels is of paramount importance to our understanding of IBC pathogenesis. Here we demonstrate that, in contrast to normal mammary epithelial cells, IBC cells evade ECM-detachment-induced apoptosis (anoikis). ErbB2 and EGFR knockdown in KPL-4 and SUM149 cells, respectively, causes decreased colony growth in soft agar and increased caspase activation following ECM detachment. ERK/MAPK signaling was found to operate downstream of ErbB2 and EGFR to protect cells from anoikis by facilitating the formation of a protein complex containing Bim-EL, LC8, and Beclin-1. This complex forms as a result of Bim-EL phosphorylation on serine 59, and thus Bim-EL cannot localize to the mitochondria and cause anoikis. These results reveal a novel mechanism that could be targeted with innovative therapeutics to induce anoikis in IBC cells.Inflammatory breast cancer (IBC) is a rare and highly invasive type of breast cancer, and patients diagnosed with IBC often face a very poor prognosis. The 5-year survival rate for patients with IBC is <40%, while the 5-year survival rate of all other breast cancers combined is approximately 90%.^{1, 2, 3, 4} This poor prognosis can be attributed to a number of factors, including the propensity for misdiagnosis of the disease due to its unique clinical presentation.^{5, 6, 7} In contrast to most breast cancers, IBC is characterized by the lack of discernible primary tumor formation and the accumulation of cancerous epithelial cells in the dermal lymphatic vessels.⁸ This lodging of IBC cells in the dermal lymphatics manifests as what appears to be inflammation, oftentimes causing clinicians to incorrectly diagnose the malady. Given that IBC cells are inherently aggressive, misdiagnosis is particularly problematic as a correct diagnosis or appropriate treatment is prolonged until more advanced disease is discovered. Thus it is imperative to gain a better understanding of the unique molecular mechanisms underlying IBC pathogenesis so that improved therapies can be designed to specifically eliminate IBC cells in a manner that improves patient outcome.Unfortunately, few treatment options exist that are specifically designed to combat IBC. A review of nearly 400 IBC patients treated at The University of Texas MD Anderson Cancer Center between 1974 and 2005 demonstrated that there has been no significant improvement in prognosis for patients with IBC over the past 30 years.¹ Many recent studies have focused on assessing the efficacy of chemotherapeutic regimens in IBC cells/patients where success had previously been observed only in the treatment of non-IBCs.^{9, 10} Some progress has been made in understanding the mechanisms underlying the invasive nature of IBC. For instance, Akt1 has been identified as a possible chemotherapeutic target that appears to be involved in the aggressive behavior of IBC cells.¹¹ Other studies have identified RhoC, which is overexpressed in 90% of IBC tissue samples, as a potent oncogene contributing to IBC pathogenesis.^{11, 12, 13, 14, 15} More recently, evidence implicating the membrane protein TIG1 and the receptor tyrosine kinase Axl in the oncogenic behavior of IBC cells has been uncovered.¹⁶ However, despite these advances, knowledge of the biological mechanisms underlying IBC pathogenesis remains fairly rudimentary, and additional research dedicated to understanding the unique molecular pathways involved in IBC progression remains essential.Given that IBC cells do not form a palpable primary tumor and instead flourish in suspension in the lymph of the dermal lymphatic vessels, we hypothesized that IBC cells must have an inherent ability to survive in the absence of attachment to the extracellular matrix (ECM). Normal mammary epithelial cells require attachment to the ECM to inhibit anoikis, which is defined as caspase-dependent cell death caused by ECM detachment.¹⁷ It has become clear that tumor progression and metastasis require cancer cells to inhibit anoikis, oftentimes through alterations in intracellular signaling pathways.^{18, 19, 20} Interestingly, previous studies have shown that ErbB2 and EGFR, which are hyperactivated in a substantial percentage of IBC patients,²¹ can effectively antagonize the anoikis program to facilitate anchorage-independent growth.^{22, 23, 24, 25, 26, 27, 28} However, a detailed examination of the molecular mechanisms underlying anoikis inhibition in IBC cells has yet to be completed. In this study, we demonstrate that signaling from EGFR and ErbB2 through ERK/MAPK has a major role in the ability of IBC cells to survive in the absence of ECM attachment. Surprisingly, we have discovered that ERK-mediated anoikis suppression in IBC cells is not due to targeting of the pro-apoptotic protein Bim-EL for degradation that has previously been observed in mammary epithelial cells.^{23, 27} Rather, ERK activation in IBC cells promotes the formation of a protein complex containing Bim-EL, Beclin-1, and LC8, which functions to sequester Bim-EL from the mitochondria and thereby block anoikis. In support of the importance of this signaling pathway in IBC patients, five of the seven IBC patient samples assayed showed discernible Bim-EL staining. Collectively, these data identify a novel mechanism utilized by IBC cells to survive during ECM detachment and reveal a potential target for the development of anoikis-inducing chemotherapeutics targeting IBC cells.     

Apoptosis induction by MEK inhibition in human lung cancer cells is mediated by Bim

AZD6244 (ARRY-142886) is an inhibitor of MEK1/2 and can inhibit cell proliferation or induce apoptosis in a cell-type dependent manner. The precise molecular mechanism of AZD6244-induced apoptosis is not clear. To investigate mechanisms of AZD6244 induced apoptosis in human lung cancer, we determined the molecular changes of two subgroups of human lung cancer cell lines that are either sensitive or resistant to AZD6244 treatment. We found that AZD6244 elicited a large increase of Bim proteins and a smaller increase of PUMA and NOXA proteins, and induced cell death in sensitive lung cancer cell lines, but had no effect on other Bcl-2 related proteins in those cell lines. Knockdown of Bim by siRNA greatly increased the IC(50) and reduced apoptosis for AZD6244 treated cells. We also found that levels of endogenous p-Thr32-FOXO3a and p-Ser253-FOXO3a were lower in AZD6244-sensitive cells than in AZD6244-resistant cells. In the sensitive cells, AZD6244 induced FOXO3a nuclear translocation required for Bim activation. Moreover, the silencing of FOXO3a by siRNA abrogated AZD6244-induced cell apoptosis. In addition, we found that transfection of constitutively active AKT up-regulated p-Thr32-FOXO3a and p-Ser253-FOXO3a expression and inhibited AZD6244-induced Bim expression in sensitive cells. These results show that Bim plays an important role in AZD6244-induced apoptosis in lung cancer cells and that the PI3K/AKT/FOXO3a pathway is involved in Bim regulation and susceptibility of lung cancer cells to AZD6244. These results have implications in the development of strategies to overcome resistance to MEK inhibitors.     

Overexpression of FGF9 in colon cancer cells is mediated by hypoxia-induced translational activation

Human fibroblast growth factor 9 (FGF9) is a potent mitogen involved in many physiological processes. Although FGF9 messenger RNA (mRNA) is ubiquitously expressed in embryos, FGF9 protein expression is generally low and restricted to a few adult organs. Aberrant expression of FGF9 usually results in human malignancies including cancers, but the mechanism remains largely unknown. Here, we report that FGF9 protein, but not mRNA, was increased in hypoxia. Two sequence elements, the upstream open reading frame (uORF) and the internal ribosome entry site (IRES), were identified in the 5'' UTR of FGF9 mRNA. Functional assays indicated that FGF9 protein synthesis was normally controlled by uORF-mediated translational repression, which kept the protein at a low level, but was upregulated in response to hypoxia through a switch to IRES-dependent translational control. Our data demonstrate that FGF9 IRES functions as a cellular switch to turn FGF9 protein synthesis ‘on’ during hypoxia, a likely mechanism underlying FGF9 overexpression in cancer cells. Finally, we provide evidence to show that hypoxia-induced translational activation promotes FGF9 protein expression in colon cancer cells. Altogether, this dynamic working model may provide a new direction in anti-tumor therapies and cancer intervention.     

Endothelin-induced prostacyclin production in rat aortic endothelial cells is mediated by protein kinase C.

Endothelin (ET) is a vasoconstrictor peptide released from endothelial cells that is known to cause prostaglandin (PG) release. The mechanism remains unclear. To determine whether the protein kinase C (PKC) signaling pathway is stimulated by endothelin, we pretreated rat aortic endothelial cells with either PKC activator or inhibitors and measured the release of prostacyclin (PGI2) by radioimmunoassay. ET (10(-9) M) produced a 10-fold increase in PGI2 release. Pretreatment with 10(-9) M of three different PKC inhibitors: 1-(5-isoquinolinesulfonyl) piperazine (CL), staurosporine, and 1-(5-isoquinolinesulfonyl-methyl) piperazine (H7) blocked ET induced PGI2 release. ET induced prostacyclin release was also blocked by pretreatment with inhibitors of either phospholipase A2 (7,7,dimethyleicosadienoic acid or trifluoromethyl ketone analogue) (10(-9) M) or cyclooxygenase (indomethacin) (10(-9) M). We conclude that ET activates PKC which activates phospholipase A2 which liberates arachidonic acid which increases PGI2 production and release.     

Activation of mitochondrial-driven apoptosis in skeletal muscle cells is not mediated by reactive oxygen species production

While the acquisition of apoptosis resistance is part of the differentiation program of skeletal muscle cells, differentiated muscle cells can undergo apoptosis in response to physiological or pathological stimuli. The generation of reactive oxygen species by mitochondria plays a major role in the control of apoptosis in many cell types. Indeed their involvement in controlling apoptosis in differentiated muscle cells, or in generating resistance to apoptosis remains unknown. Moreover, differentiated muscle cells specifically express the uncoupling protein-3, a mitochondrial protein potentially involved in controlling reactive oxygen species production. To study the role of mitochondrial reactive oxygen species in the control of apoptosis in skeletal muscle cells, L6E9 myoblasts and myotubes were exposed to staurosporine, an inducer of apoptosis via mitochondrial pathways. Staurosporine activated apoptotic pathways (i.e. caspase-3 and caspase-9) increasing reactive oxygen species in myoblasts and, to a minor extent, in myotubes. However, the increase in reactive oxygen species was not needed to induce apoptosis nor was it involved in the differential sensitization of myoblasts and myotubes to apoptosis. Moreover, expression of uncoupling protein-3 in myotubes did not affect reactive oxygen species production, although it produced a slight sensitization for staurosporine-induced apoptosis. Results indicate that apoptotic activation in skeletal muscle cells mainly involves reactive oxygen species-independent mechanisms and that mitochondrial uncoupling protein-3 is not protective either for reactive oxygen species production or for apoptotic activation in muscle cells.     

Withaferin A-induced apoptosis in human breast cancer cells is mediated by reactive oxygen species

Withaferin A (WA), a promising anticancer constituent of Ayurvedic medicinal plant Withania somnifera, inhibits growth of MDA-MB-231 and MCF-7 human breast cancer cells in culture and MDA-MB-231 xenografts in vivo in association with apoptosis induction, but the mechanism of cell death is not fully understood. We now demonstrate, for the first time, that WA-induced apoptosis is mediated by reactive oxygen species (ROS) production due to inhibition of mitochondrial respiration. WA treatment caused ROS production in MDA-MB-231 and MCF-7 cells, but not in a normal human mammary epithelial cell line (HMEC). The HMEC was also resistant to WA-induced apoptosis. WA-mediated ROS production as well as apoptotic histone-associated DNA fragment release into the cytosol was significantly attenuated by ectopic expression of Cu,Zn-superoxide dismutase in both MDA-MB-231 and MCF-7 cells. ROS production resulting from WA exposure was accompanied by inhibition of oxidative phosphorylation and inhibition of complex III activity. Mitochondrial DNA-deficient Rho-0 variants of MDA-MB-231 and MCF-7 cells were resistant to WA-induced ROS production, collapse of mitochondrial membrane potential, and apoptosis compared with respective wild-type cells. WA treatment resulted in activation of Bax and Bak in MDA-MB-231 and MCF-7 cells, and SV40 immortalized embryonic fibroblasts derived from Bax and Bak double knockout mouse were significantly more resistant to WA-induced apoptosis compared with fibroblasts derived from wild-type mouse. In conclusion, the present study provides novel insight into the molecular circuitry of WA-induced apoptosis involving ROS production and activation of Bax/Bak.     

8-Isoprostane-induced endothelin-1 production by infant rat pulmonary artery smooth muscle cells is mediated by Rho-kinase

We have reported that 8-isoprostane stimulated the production of endothelin (ET)-1, a potent vasoconstrictor and critical mediator of chronic pulmonary hypertension, by infant rat pulmonary artery smooth muscle cells (PASMCs), through stimulation of the thromboxane A2 receptor. The aim of this study was to examine the contribution of putative downstream intracellular mediators of thromboxane A2 receptor stimulation to this effect. PASMCs from infant rats were treated with calcium ionophore (A23187), 8-isoprostane, or 8-isoprostane together with inhibitors of tyrosine kinase, protein kinase C, phosphatidylinositol 3-kinase, mitogen-activated protein kinases, or Rho-kinases (ROCK). A23187 had no effect on ET-1 production, excluding raised intracellular Ca2+ as a major contributor. Increased ET-1 production induced by 8-isoprostane was significantly attenuated by the ROCK inhibitors Y-27632 and hydroxyfasudil, but not by inhibitors of the other pathways. 8-Isoprostane also increased membrane binding of RhoA, a major determinant of ROCK activity, and ROCK-II expression through the protein kinase C pathway. These data indicate that the RhoA/ROCK pathway mediates increased ET-1 production by PASMCs, which we speculate may at least partly explain the beneficial effects of both antioxidants and ROCK inhibitors in animal models of chronic pulmonary hypertension.     

IL-12-independent IFN-gamma production by T cells in experimental Chagas' disease is mediated by IL-18.

IL-12p35-deficient (IL-12p35(-/-)) mice were highly susceptible to Trypanosoma cruzi infection and succumbed during acute infection, demonstrating the crucial importance of endogenous IL-12 in resistance to experimental Chagas' disease. Delayed immune responses were observed in mutant mice, although comparable IFN-gamma and TNF-alpha blood levels as in wild-type mice were detected 2 wk postinfection. In vivo and in vitro analysis demonstrated that T cells, but not NK cells, were recruited to infected organs. Analysis of mice double deficient in the recombinase-activating gene 2 (RAG2) and IL-12p35, as well as studies involving T cell depletion, identified CD4(+) T cells as the cellular source for IL-12-independent IFN-gamma production. IL-18 was induced in IL-12p35(-/-) mice and was responsible for IFN-gamma production, as demonstrated by in vivo IL-18 neutralization studies. In conclusion, evidence is presented for an IL-12-independent IFN-gamma production in experimental Chagas' disease that is T cell and IL-18 dependent.     

CRH stimulation of corticosteroids production in melanocytes is mediated by ACTH

The response to systemic stress is organized along the hypothalamic-pituitary-adrenal axis (HPA), whereas the response to a peripheral stress (solar radiation) is mediated by epidermal melanocytes (cells of neural crest origin) responsible for the pigmentary reaction. Melanocytes express proopiomelanocortin (POMC), corticotropin-releasing hormone (CRH), and CRH receptor-1 (CRH-R1) and can produce corticosterone. In the present study, incubation of normal epidermal melanocytes with CRH was found to trigger a functional cascade structured hierarchically and arranged along the same algorithm as in the HPA axis: CRH activation of CRH-R1 stimulated cAMP accumulation and increased POMC gene expression and production of ACTH. CRH and ACTH also enhanced production of cortisol and corticosterone, and cortisol production was also stimulated by progesterone. The chemical identity of the cortisol was confirmed by liquid chromatography-mass spectrometry (LC/MS2) with [corrected] mass spectrometry-mass spectrometry analyses. POMC gene silencing abolished the stimulatory effect of CRH on corticosteroid synthesis, indicating that this is indirect and mediated via production of ACTH. Thus the melanocyte response to CRH is highly organized along the same functional hierarchy as the HPA axis. This pattern demonstrates the fractal nature of the response to stress with similar activation sequence at the single-cell and whole body levels.     

Plumbagin-induced apoptosis of human breast cancer cells is mediated by inactivation of NF-kappaB and Bcl-2

Breast cancer remains the major cause of cancer-related deaths in women world-wide. The heterogeneity of breast cancer has further complicated the progress of target-based therapies. Triple negative breast cancers, lacking estrogen receptor, progesterone receptor and the Her-2/neu (ErbB2), represent a highly aggressive breast cancer subtype, that are difficult to treat. Pleiotropic agents, such as those found in nature, can target receptor-positive as well as receptor-negative cancer cells, suggesting that such agents could have significant impact in breast cancer prevention and/or therapy. Plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone) is one such agent which has anti-tumor activity against several cancers. However, its mechanism of action against breast cancer is not clearly understood. We hypothesized that plumbagin may act as an effective agent against breast cancer especially triple negative breast cancer. We tested our hypothesis using ER-positive MCF-7 and ER-negative MDA-MB-231 (triple negative) breast cancer cells, and we found that plumbagin significantly inhibits the growth of breast cancer cells with no effect on normal breast epithelial cells. We also found that plumbagin induces apoptosis with concomitant inactivation of Bcl-2 and the DNA binding activity of NF-kappaB. Bcl-2 over-expression resulted in attenuation of plumbagin-induced effects, suggesting that the inhibition of cell growth and induction of apoptosis by plumbagin is in part due to inactivation of NF-kappaB/Bcl-2 pathway. To our knowledge, this is the first report, showing mechanistic and cancer cell specific apoptosis-inducing effects of plumbagin in breast cancer cells, suggesting the potential role of plumbagin in the prevention and/or treatment of breast cancer.     

Desensitization of inositol phosphate production after agonist stimulation of endothelial cells is not mediated by protein kinase C

To investigate the possible role of protein kinase C activation in the desensitization of inositol phosphate production in endothelial cells we compared desensitization induced by agonists to that induced by the phorbol ester TPA. While histamine or thrombin induced desensitization of inositol phosphate production is homologous TPA induced desensitization is heterologous. The protein kinase C inhibitor H-7 reduced TPA desensitization but had no effect on the agonist induced desensitization. While downregulation of protein kinase C by long term (24 hr) treatment of the cells with TPA reduced the desensitization mediated by short term TPA-treatment it did not affect the agonist induced desensitization. These results suggest that desensitization of inositol phosphate production after agonist stimulation of endothelial cells is not mediated by protein kinase C.     

Serum deprivation-induced reactive oxygen species production is mediated by Romo1

Serum deprivation-triggered increases in reactive oxygen species (ROS) are known to induce apoptotic cell death. However, the mechanism by which serum deprivation causes ROS production is not known. Since mitochondria are the main source of ROS and since mitochondrial ROS modulator 1 (Romo1) is involved in ROS production, we sought to determine if serum deprivation triggered ROS production through Romo1. To examine the relationship between Romo1 and the serum deprivation-triggered increase in ROS, we transfected Romo1 siRNA into various cell lines and looked for inhibition of mitochondrial ROS generation. Romo1 knockdown by Romo1 siRNA blocked the mitochondrial ROS production caused by serum deprivation, which originates in the mitochondrial electron transport chain. We also found that Romo1 knockdown inhibited serum deprivation-induced apoptosis. These findings suggest that Romo1-derived ROS play an important role in apoptotic cell death triggered by withdrawal of cell survival factors.     

Proliferation of AXC/SSh rat prostate cancer cells in vitro is androgen modulated

We used heterogeneous parental cultures of AXC/SSh rat prostate cancer cells to isolate clonally derived prostate cancer cell lines. Light and electron microscopic analyses established that parental and clonally isolated cells possess features characteristic of secretory epithelium. Biochemical analyses showed that these cells contained androgen receptors and acid phosphatase and 5 alpha-reductase activity; phenotypic markers characteristic of differentiated prostate epithelium. Content of these prostate epithelial cell markers was variable and cell line specific. We used selected cell lines to examine androgen modulation of AXC/SSh rat prostate cancer cell proliferation in vitro. We found that proliferation of C-family or D-family cells, those respectively maintained on medium without additions or medium containing 10(-7) M 5 alpha-dihydrotestosterone, was not affected by changes in medium testosterone concentration through the range 10(-6)-10(-9) M. In contrast, testosterone modified proliferation of T-family cells, those maintained on medium containing 10(-7) M testosterone, and effects were antagonized by the anti-androgen RU 23908. Preliminary studies established that AXC/SSh rat prostate cancer cells elaborate polypeptide components which stimulate in vitro cell proliferation. Both the ability to elaborate these components and their effects on in vitro cell proliferation appeared to be cell line specific.     

Endotoxin-induced zinc accumulation by liver cells is mediated by metallothionein synthesis

Endotoxin induces a decrease in zinc concentration in the serum and an increase in zinc levels in the liver. We have studied whether metallothionein (MT), which is a heavy metal-binding protein, is associated with this phenomenon in vitro. When MT of liver cells is induced by a factor secreted by endotoxin-stimulated macrophages, the cells accumulate zinc from the medium. The temporal accumulation of zinc is correlated with the induction of MT, and the accumulated zinc binds to MT. These results suggest that zinc accumulation by liver cells is mediated by metallothionein produced in response to a macrophage factor, which is elicited by endotoxin.     

Kaempferol-induced growth inhibition and apoptosis in A549 lung cancer cells is mediated by activation of MEK-MAPK

A vast variety of naturally occurring substances have been shown to protect against experimental carcinogenesis and an increasing amount of evidence suggests that kaempferol may have cancer chemopreventative properties. However, the precise underlying protective mechanisms are poorly understood. To elucidate these mechanisms, we challenged human lung cancer cell line A549 with kaempferol and investigated its effects upon cellular growth and signal transduction pathways. Treatment of A549 cells with kaempferol resulted in a dose- and time-dependent reduction in cell viability and DNA synthesis with the rate of apoptosis equivalent to 0.9+/-0.5, 5.2+/-1.5, 16.8+/-2.0, 25.4+/-2.6, and 37.8+/-4.5% on treatment with 0, 17.5, 35.0, 52.5, and 70.0 microM kaempferol, respectively. Concomitantly, kaempferol treatments led to a 1.2-, 2.7-, 3.3-, and 3.4-fold increase in Bax. Similar elevations were also observed in Bad which increased 1.2-, 3.3-, 3.7-, and 4.7-fold, respectively, as compared to control. Bcl-2 and Bcl-xL expression were inhibited in a dose-dependent fashion. While the Akt-1 and phosphorylated Akt-1 were inhibited, the mitogen-activated protein kinase (MAPK) was activated upon kaempferol treatment. Kaempferol induced apoptosis was associated with the cleavage of caspase-7 and poly ADP-ribose polymerase (PARP). Inhibition of MEK1/2 but not PI-3 kinase blocked kaempferol-induced cleavage of caspase-7, PARP cleavage, and apoptosis. The results suggest that inactivation of Akt-1 and alteration of Bcl-2 family of proteins are not sufficient for kaempferol to induce apoptosis and activation of MEK-MAPK is a requirement for kaempferol-induced cell death machinery in A549 cells.