Vascular disrupting agents (VDAs) represent a novel approach to the treatment of cancer, resulting in the collapse of tumor vasculature and tumor death. 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a VDA currently in advanced phase II clinical trials, yet its precise mechanism of action is unknown despite extensive preclinical and clinical investigations. Our data demonstrate that DMXAA is a novel and specific activator of the TANK-binding kinase 1 (TBK1)–interferon (IFN) regulatory factor 3 (IRF-3) signaling pathway. DMXAA treatment of primary mouse macrophages resulted in robust IRF-3 activation and ∼750-fold increase in IFN-β mRNA, and in contrast to the potent Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS), signaling was independent of mitogen-activated protein kinase (MAPK) activation and elicited minimal nuclear factor κB–dependent gene expression. DMXAA-induced signaling was critically dependent on the IRF-3 kinase, TBK1, and IRF-3 but was myeloid differentiation factor 88–, Toll–interleukin 1 receptor domain–containing adaptor inducing IFN-β–, IFN promoter-stimulator 1–, and inhibitor of κB kinase–independent, thus excluding all known TLRs and cytosolic helicase receptors. DMXAA pretreatment of mouse macrophages induced a state of tolerance to LPS and vice versa. In contrast to LPS stimulation, DMXAA-induced IRF-3 dimerization and IFN-β expression were inhibited by salicylic acid. These findings detail a novel pathway for TBK1-mediated IRF-3 activation and provide new insights into the mechanism of this new class of chemotherapeutic drugs.
DMXAA (5,6-dimethylxanthenone-4-acetic acid), a novel anti-tumour agent currently undergoing clinical evaluation, appears to mediate its anti-tumour effects through immune modulation and the production of the cytokine tumour necrosis factor-alpha (TNF). Our previous studies have shown that thalidomide, a potent inhibitor of TNF biosynthesis that has numerous biological effects, including inhibition of tumour angiogenesis, unexpectedly augments the anti-tumour response in mice to DMXAA. We show here that thalidomide (100 mg kg(-1)) has no effect when administered with inactive doses of DMXAA, and that it must be given simultaneously with an active dose of DMXAA to have its maximum potentiating effect on the growth of the murine Colon 38 adenocarcinoma. To address the issue of whether inhibition of serum TNF production is important for potentiation of anti-tumour activity, we have tested three potent analogues of thalidomide. All three analogues, when co-administered with DMXAA to mice at doses lower than those used with thalidomide, inhibited TNF production and were effective in potentiating the anti-tumour activity of DMXAA against transplanted Colon 38 tumours. One of the analogues, N-phenethyltetrafluorophthalimide, was 1000-fold more potent than thalidomide and at a dose of 0.1 mg kg(-1) in combination with DMXAA (30 mg kg(-1)) cured 100% of mice, compared with 67% for the group treated with DMXAA alone. We also tested pentoxifylline and found it to suppress TNF production in response to DMXAA and to potentiate the anti-tumour effect of DMXAA. The results are compatible with the hypothesis that pharmacological reduction of serum TNF levels might benefit the anti-tumour effects of DMXAA and suggest new strategies for therapy using this agent.
The signaling pathway(s) and molecular target(s) for 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a tumor vascular disrupting agent in late stages of clinical development, are still undefined. As an approach toward identifying potential targets for DMXAA, a tritiated azido-analog of DMXAA was used to probe for cellular binding proteins. More than 20 cytosolic proteins from murine splenocytes, RAW 264.7 cells, and the HECPP immortalized endothelial cells were photoaffinity-labeled. Although no protein domain, fold, or binding site for a specific ligand was found to be shared by all the candidate proteins, essentially all were noted to be oxidizable proteins, implicating a role for redox signaling in the action of DMXAA. Consistent with this hypothesis, DMXAA caused an increase in concentrations of reactive oxygen species (ROS) in RAW264.7 cells during the first 2 hours. This increase in ROS was suppressed in the presence of the antioxidant, N-acetyl-l-cysteine, which also suppressed DMXAA-induced cytokine production in the RAW 264.7 cells with no effects on cell viability. Short interfering RNA (siRNA)-mediated knockdown of one of the photoaffinity-labeled proteins, superoxide dismutase 1, an ROS scavenger, resulted in an increase in tumor necrosis factor-α production by RAW 264.7 cells in response to DMXAA compared with negative or positive controls transfected with nontargeting or lamin A/C-targeting siRNA molecules, respectively. The results from these lines of study all suggest that redox signaling plays a central role in cytokine induction by DMXAA.
5,6-Dimethylxanthenone-4-acetic acid (DMXAA) acts through tumor vascular disruption and cytokine production and is the first of its class to enter phase 3 trials. We characterized leukocytes and cytokines in murine Colon 38 tumors before and after DMXAA treatment. Tumor mass declined 50% 24 hours after DMXAA administration, but the leukocyte count per gram of tumor increased threefold owing to a large influx of Ly6G+CD11b+F4/80- cells with the morphology of neutrophils. However, B and T lymphocytes, natural killer cells, and macrophages in the tumor all decreased in numbers. Seven chemokines were substantially induced in the tumor, spleen, and serum 4 hours after DMXAA administration. Using cultured spleen cell subpopulations, CD11b+ cells (largely monocytes and macrophages) were shown to be the primary producers of tumor necrosis factor α, interleukin 6 (IL-6), and macrophage inflammatory 1α (MIP-1α). CD49b+ natural killer cells produced IP-10, whereas CD45R+ B lymphocytes produced regulated upon activation normal T cell express sequence. T lymphocytes were not major producers of cytokines in the response to DMXAA. Murine peripheral blood leukocytes (PBLs) produced a similar panel of cytokines in culture to that detected in mouse serum after DMXAA treatment. Cytokines in human PBL cultures were subsequently measured with the aim of identifying potential serum markers of the human response to DMXAA. IP-10 (P < .001), monocyte chemoattractant protein 1 (P < .001), and sCD40L (P < .01) were decreased, whereas IL-8 (P < .001) and MIP-1α (P = .03) were increased in DMXAA-treated compared with untreated PBL cultures from a group of 12 donors.
Mitogen-activated protein kinases (MAPKs) are serine-threonine protein kinases that play the major role in signal transduction from the cell surface to the nucleus. MAPKs, which consist of growth factor-regulated extracellular signal-related kinases (ERKs), and the stress-activated MAPKs, c-jun NH2-terminal kinases (JNKs) and p38 MAPKs, are part of a three-kinase signaling module composed of the MAPK, an MAPK kinase (MAP2K) and an MAPK kinase (MAP3K). MAP3Ks phosphorylate MAP2Ks, which in turn activate MAPKs. MAPK phosphatases (MKPs), which recognize the TXY amino acid motif present in MAPKs, dephosphorylate and deactivate MAPKs. MAPK pathways are known to be influenced not only by receptor ligand interactions, but also by different stressors placed on the cell. One type of stress that induces potential activation of MAPK pathways is the oxidative stress caused by reactive oxygen species (ROS). Generally, increased ROS production in a cell leads to the activation of ERKs, JNKs, or p38 MAPKs, but the mechanisms by which ROS can activate these kinases are unclear. Oxidative modifications of MAPK signaling proteins and inactivation and/or degradation of MKPs may provide the plausible mechanisms for activation of MAPK pathways by ROS, which will be reviewed in this paper.
The tumor-vascular disrupting agent (VDA) vadimezan (5,6-dimethylxanthenone-4-acetic acid, DMXAA) has been shown to potentiate the antitumor activity of photodynamic therapy (PDT) using systemically administered photosensitizers. Here, we characterized the response of subcutaneous syngeneic Colon26 murine colon adenocarcinoma tumors to PDT using the locally applied photosensitizer precursor aminolevulinic acid (ALA) in combination with a topical formulation of vadimezan. Diffuse correlation spectroscopy (DCS), a non-invasive method for monitoring blood flow, was utilized to determine tumor vascular response to treatment. Additionally, correlative CD31-immunohistochemistry to visualize endothelial damage, ELISA assays to measure induction of tumor necrosis factor-alpha (TNF-α) and tumor weight measurements were also examined in separate animals. In our previous work, DCS revealed a selective decrease in tumor blood flow over time following topical vadimezan. ALA-PDT treatment also induced a decrease in tumor blood flow. The onset of blood flow reduction was rapid in tumors treated with both ALA-PDT and vadimezan. CD31-immunostaining of tumor sections confirmed vascular damage following topical application of vadimezan. Tumor weight measurements revealed enhanced tumor growth inhibition with combination treatment compared to ALA-PDT or vadimezan treatment alone. In conclusion, vadimezan as a topical agent enhances treatment efficacy when combined with ALA-PDT. This combination could be useful in clinical applications.
Analyses of mitogen-activated protein kinases (MAPKs) in a mouse hepatitis virus (MHV)-infected macrophage-derived J774.1 cell line showed activation of two MAPKs, p38 MAPK and c-Jun N-terminal kinase (JNK), but not of extracellular signal-regulated kinase (ERK). Activation of MAPKs was evident by 6 h postinfection. However, UV-irradiated MHV failed to activate MAPKs, which demonstrated that MHV replication was necessary for their activation. Several other MHV-permissive cell lines also showed activation of both p38 MAPK and JNK, which indicated that the MHV-induced stress-kinase activation was not restricted to any particular cell type. The upstream kinase responsible for activating MHV-induced p38 MAPK was the MAPK kinase 3. Experiments with a specific inhibitor of p38 MAPK, SB 203580, demonstrated that MHV-induced p38 MAPK activation resulted in the accumulation of interleukin-6 (IL-6) mRNAs and an increase in the production of IL-6, regardless of MHV-induced general host protein synthesis inhibition. Furthermore, MHV production was suppressed in SB 203580-treated cells, demonstrating that activated p38 MAPK played a role in MHV replication. The reduced MHV production in SB 203580-treated cells was, at least in part, due to a decrease in virus-specific protein synthesis and virus-specific mRNA accumulation. Interestingly, there was a transient increase in the amount of phosphorylation of the translation initiation factor 4E (eIF4E) in infected cells, and this eIF4E phosphorylation was p38 MAPK dependent; it is known that phosphorylated eIF4E enhances translation rates of cap-containing mRNAs. Furthermore, the upstream kinase responsible for eIF4E phosphorylation, MAPK-interacting kinase 1, was also phosphorylated and activated in response to MHV infection. Our data suggested that host cells, in response to MHV replication, activated p38 MAPK, which subsequently phosphorylated eIF4E to efficiently translate certain host proteins, including IL-6, during virus-induced severe host protein synthesis inhibition. MHV utilized this p38 MAPK-dependent increase in eIF4E phosphorylation to promote virus-specific protein synthesis and subsequent progeny virus production. Enhancement of virus-specific protein synthesis through virus-induced eIF4E activation has not been reported in any other viruses.
The tumour blood flow inhibitor 5,6-dimethylxanthenone-4-acetic acid (DMXAA) causes dramatic haemorrhagic necrosis in murine tumours, but activity is seen only at doses close to the toxic limit. This study investigates two approaches for increasing the therapeutic ratio of DMXAA. The first approach combines DMXAA with a second tumour blood flow inhibitor, 5-hydroxytryptamine (5-HT). Co-administration of 5-HT (700 micromol kg(-1)) to C3H mice caused marked enhancement of DMXAA effects against MDAH-MCa-4 tumours, with dose-modifying factors (DMFs) of >3 for blood flow inhibition (at 4 h), 2.3 for necrosis (at 12 h) and 2.0 for growth delay, without compromising the maximum tolerated dose of DMXAA (90 micromol kg(-1)). The data are consistent with ischaemic injury to the tumour being the major mechanism of anti-tumour activity. The second approach combines DMXAA (+/- 5-HT) with hypoxia-selective bioreductive drugs. Anti-tumour activity of all three bioreductive drugs tested (tirapazamine, CI-1010, SN 23816) was strongly potentiated by DMXAA, suggesting that there is a population of reversibly hypoxic tumour cells after DMXAA treatment. Co-administration of 5-HT further potentiated anti-tumour activity, but also increased host toxicity of tirapazamine and CI-1010 so that little therapeutic benefit was achieved. In contrast, the host toxicity of the dinitrobenzamide mustard SN 23816 was only slightly increased by DMXAA/5-HT, whereas the tumour growth delay at the maximum tolerated dose of SN 23816 was increased from 3.5 to 26.5 days. This study demonstrates that 5-HT and/or bioreductive drugs can improve the therapeutic activity of DMXAA in mice, and that with SN 23816 both approaches can be used together to provide considerably enhanced anti-tumour activity.
5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a potent type I interferon (IFN) inducer, was evaluated as a chemotherapeutic agent in mouse cancer models and proved to be well tolerated in human cancer clinical trials. Despite its multiple biological functions, DMXAA has not been fully characterized for the potential application as a vaccine adjuvant. In this report, we show that DMXAA does act as an adjuvant due to its unique property as a soluble innate immune activator. Using OVA as a model antigen, DMXAA was demonstrated to improve on the antigen specific immune responses and induce a preferential Th2 (Type-2) response. The adjuvant effect was directly dependent on the IRF3-mediated production of type-I-interferon, but not IL-33. DMXAA could also enhance the immunogenicity of influenza split vaccine which led to significant increase in protective responses against live influenza virus challenge in mice compared to split vaccine alone. We propose that DMXAA can be used as an adjuvant that targets a specific innate immune signaling pathway via IRF3 for potential applications including vaccines against influenza which requires a high safety profile.
The adverse effects of lipopolysaccharide (LPS) are mediated primarily by tumor necrosis factor alpha (TNF-alpha). TNF-alpha production by LPS-stimulated macrophages is regulated at the levels of both transcription and translation. It has previously been shown that several mitogen-activated protein kinases (MAPKs) are activated in response to LPS. We set out to determine which MAPK signaling pathways are activated in our system and which MAPK pathways are required for TNF-alpha gene transcription or TNF-alpha mRNA translation. We confirm activation of the MAPK family members extracellular-signal-regulated kinases 1 and 2 (ERK1 and ERK2), p38, and Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), as well as activation of the immediate upstream MAPK activators MAPK/ERK kinases 1 and 4 (MEK1 and MEK4). We demonstrate that LPS also activates MEK2, MEK3, and MEK6. Furthermore, we demonstrate that dexamethasone, which inhibits the production of cytokines, including TNF-alpha, significantly inhibits LPS induction of JNK/SAPK activity but not that of p38, ERK1 and ERK2, or MEK3, MEK4, or MEK6. Dexamethasone also blocks the sorbitol but not anisomycin stimulation of JNK/SAPK activity. A kinase-defective mutant of SAPKbeta, SAPKbeta K-A, blocked translation of TNF-alpha, as determined by using a TNF-alpha translational reporting system. Finally, overexpression of wild-type SAPKbeta was able to overcome the dexamethasone-induced block of TNF-alpha translation. These data confirm that three MAPK family members and their upstream activators are stimulated by LPS and demonstrate that JNK/SAPK is required for LPS-induced translation of TNF-alpha mRNA. A novel mechanism by which dexamethasone inhibits translation of TNF-alpha is also revealed.
Elevated systemic levels of pro-inflammatory cytokines cause hypotension during septic shock and induce capillary leakage in acute lung injury. Manassantin B has anti-inflammatory and anti-plasmoidal properties. This study examined the effects of manassantin B on lipopolysaccharide (LPS)-induced inflammatory response in murine macrophages.
RAW 264.7 macrophage cells were incubated without or with (1, 3 and 10 µM) manassantin B and without or with (100 ng/ml) LPS. Manassantin B dissolved in phosphate buffered saline was added to the medium 1 h prior to the addition of LPS. The degree of activation of mitogen-activated protein kinase (MAPK) including extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino terminal kinases (JNK) and p38 MAPK, and the level of interleukin (IL)-1β were determined 30 min and 24 h after the addition of LPS respectively.
Manassantin B inhibited the production of IL-1β and attenuated the phosphorylations of ERK1/2 and p38 MAPK, but not that of JNK, in RAW 264.7 cells treated with LPS.
Manassantin B reduces LPS-induced IL-1β expression through effects on ERK1/2- and p38 MAPK-mediated pathways. Manassantin B has potential as a potent anti-inflammatory drug for use in pathological processes such as sepsis or acute lung injury.
Interleukin-1β; Lipopolysaccharides; Manassantin B; Mitogen-activated protein kinase
The descending pain modulatory system is thought to undergo plastic changes following peripheral tissue injury and exerts bidirectional (facilitatory and inhibitory) influence on spinal nociceptive transmission. The mitogen-activated protein kinases (MAPKs) superfamily consists of four main members: the extracellular signal-regulated protein kinase1/2 (ERK1/2), the c-Jun N-terminal kinases (JNKs), the p38 MAPKs, and the ERK5. MAPKs not only regulate cell proliferation and survival but also play important roles in synaptic plasticity and memory formation. Recently, many studies have demonstrated that noxious stimuli activate MAPKs in several brain regions that are components of descending pain modulatory system. They are involved in pain perception and pain-related emotional responses. In addition, psychophysical stress also activates MAPKs in these brain structures. Greater appreciation of the convergence of mechanisms between noxious stimuli- and psychological stress-induced neuroplasticity is likely to lead to the identification of novel targets for a variety of pain syndromes.
ERK1 and ERK2 mitogen-activated protein kinases (MAPK) play a critical role in regulation of cell proliferation and differentiation in response to mitogens and other extracellular stimuli. Mitogens and cytokines that activate MAPK in T cells have been shown to activate human immunodeficiency virus type 1 (HIV-1) replication. Little is known about the signal transduction pathways that activate HIV-1 replication in T cells upon activation by extracellular stimulation. Here, we report that activation of MAPK through the Ras/Raf/MEK signaling pathway enhances the infectivity of HIV-1 virions. Virus infectivity was enhanced by treatment of cells with MAPK stimulators, such as serum and phorbol myristate acetate, as well as by coexpression of constitutively activated Ras, Raf, or MEK (MAPK kinase) in the absence of extracellular stimulation. Treatment of cells with PD 098059, a specific inhibitor of MAPK activation, or with a MAPK antisense oligonucleotide reduced the infectivity of HIV-1 virions without significantly affecting virus production or the levels of virion-associated Gag and Env proteins. MAPK has been shown to regulate HIV-1 infectivity by phosphorylating Vif (X. Yang and D. Gabuzda, J. Biol. Chem. 273:29879–29887, 1998). However, MAPK activation enhanced virus infectivity in some cells lines that do not require Vif function. The HIV-1 Rev, Tat, p17Gag, and Nef proteins were directly phosphorylated by MAPK in vitro, suggesting that other HIV-1 proteins are potential substrates for MAPK phosphorylation. These results suggest that activation of the ERK MAPK pathway plays a role in HIV-1 replication by enhancing the infectivity of HIV-1 virions through Vif-dependent as well as Vif-independent mechanisms. MAPK activation in producer cells may contribute to the activation of HIV-1 replication when T cells are activated by mitogens and other extracellular stimuli.
Mitogen-activated protein kinases (MAPKs) comprise a family of serine/threonine kinases that are activated by a large variety of extracellular stimuli and play integral roles in controlling many cellular processes, from the cell surface to the nucleus. The MAPK family includes four distinct MAPK cascades, that is, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK, c-Jun N-terminal kinase or stress-activated protein kinase, and ERK5. These MAPKs are essentially operated through three-tiered consecutive phosphorylation events catalyzed by a MAPK kinase kinase, a MAPK kinase, and a MAPK. MAPKs lie in protein kinase cascades. The MAPK signaling pathways have been demonstrated to be associated with events regulating the expression of the steroidogenic acute regulatory protein (StAR) and steroidogenesis in steroidogenic tissues. However, it has become clear that the regulation of MAPK-dependent StAR expression and steroid synthesis is a complex process and is context dependent. This paper summarizes the current level of understanding concerning the roles of the MAPK signaling cascades in the regulation of StAR expression and steroidogenesis in different steroidogenic cell models.
Intracellular mitogen-activated protein kinase (MAPK) signaling cascades likely play an important role in the pathogenesis of cardiac and vascular disease. A substantial amount of basic science research has defined many of the details of MAPK pathway organization and activation, but the role of individual signaling proteins in the pathogenesis of various cardiovascular diseases is still being elucidated. In this review, the role of the MAPKs extracellular signal-regulated kinase (ERK), C-jun N-terminal kinase (JNK) and p38 MAPK in cardiac hypertrophy, cardiac remodeling after myocardial infarction, atherosclerosis and vascular restenosis will be examined with attention paid to genetically-modified murine model systems and to the use of pharmacologic inhibitors of protein kinases. Despite the complexities of this field of research, attractive targets for pharmacological therapy are emerging.
Renal cell carcinoma (RCC) is an angiogenesis-dependent and hypoxia-driven malignancy. As a result, there has been an increased interest in the use of antiangiogenic agents for the management of RCC in patients. However, the activity of tumor-vascular disrupting agents (tumor-VDA) has not been extensively examined against RCC. In this study, we investigated the therapeutic efficacy of the tumor-VDA ASA404 (DMXAA, 5,6-dimethylxanthenone-4-acetic acid, or vadimezan) in combination with the mTOR inhibitor everolimus (RAD001) against RCC. In vitro studies were carried out using human umbilical vein endothelial cells and in vivo studies using orthotopic RENCA tumors and immunohistochemical patient tumor-derived RCC xenografts. MRI was used to characterize the vascular response of orthotopic RENCA xenografts to combination treatment. Therapeutic efficacy was determined by tumor growth measurements and histopathologic evaluation. ASA404/everolimus combination resulted in enhanced inhibition of endothelial cell sprouting in the 3-dimensional spheroid assay. MRI of orthotopic RENCA xenografts revealed an early increase in permeability 4 hours posttreatment with ASA404, but not with everolimus. Twenty-four hours after treatment, a significant reduction in blood volume was observed with combination treatment. Correlative CD31/NG2 staining of tumor sections confirmed marked vascular damage following combination therapy. Histologic sections showed extensive necrosis and a reduction in the viable rim following combination treatment compared with VDA treatment alone. These results show the potential of combining tumor-VDAs with mTOR inhibitors in RCC. Further investigation into this novel combination strategy is warranted.
Summary: The mitogen-activated protein kinases (MAPKs) regulate diverse cellular programs by relaying extracellular signals to intracellular responses. In mammals, there are more than a dozen MAPK enzymes that coordinately regulate cell proliferation, differentiation, motility, and survival. The best known are the conventional MAPKs, which include the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino-terminal kinases 1 to 3 (JNK1 to -3), p38 (α, β, γ, and δ), and ERK5 families. There are additional, atypical MAPK enzymes, including ERK3/4, ERK7/8, and Nemo-like kinase (NLK), which have distinct regulation and functions. Together, the MAPKs regulate a large number of substrates, including members of a family of protein Ser/Thr kinases termed MAPK-activated protein kinases (MAPKAPKs). The MAPKAPKs are related enzymes that respond to extracellular stimulation through direct MAPK-dependent activation loop phosphorylation and kinase activation. There are five MAPKAPK subfamilies: the p90 ribosomal S6 kinase (RSK), the mitogen- and stress-activated kinase (MSK), the MAPK-interacting kinase (MNK), the MAPK-activated protein kinase 2/3 (MK2/3), and MK5 (also known as p38-regulated/activated protein kinase [PRAK]). These enzymes have diverse biological functions, including regulation of nucleosome and gene expression, mRNA stability and translation, and cell proliferation and survival. Here we review the mechanisms of MAPKAPK activation by the different MAPKs and discuss their physiological roles based on established substrates and recent discoveries.
The acute effects of the vascular-disrupting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) were investigated in vivo using intravital microscopy (IVM) and magnetic resonance imaging (MRI). Changes in vascular permeability and blood flow of syngeneic CT-26 murine colon adenocarcinomas were assessed at 4 and 24 hours after DMXAA treatment (30 mg/kg, i.p.) and correlated with induction of tumor necrosis factor-α (TNF-α), endothelial damage [CD31/terminal deoxynucleotidyl transferase (TdT)], and treatment outcome. Intravital imaging revealed a marked increase in vascular permeability 4 hours after treatment, consistent with increases in intratumoral mRNA and protein levels of TNF-α. Parallel contrast-enhanced MRI studies showed a ∼ 4-fold increase in longitudinal relaxation rates (ΔR1), indicative of increased contrast agent accumulation within the tumor. Dual immunostained tumor sections (CD31/TdT) revealed evidence of endothelial apoptosis at this time point. Twenty-four hours after treatment, extensive hemorrhage and complete disruption of vascular architecture were observed with IVM, along with a significant reduction in ΔR1; and virtual absence of CD31 immunostaining. DMXAA-induced tumor vascular damage resulted in significant long-term (60-day) cures compared to untreated controls. Multimodality imaging approaches are useful in visualizing the effects of antivascular therapy in vivo. Such approaches allow cross validation and correlation of findings with underlying molecular changes contributing to treatment outcome.
Vascular-disrupting agents; DMXAA; multimodality imaging; tumor vasculature; tumor necrosis factor-α
Idiopathic pulmonary fibrosis is a chronic lung disorder characterized by fibroblasts proliferation and extracellular matrix accumulation. Induction of fibroblast apoptosis therefore plays a crucial role in the resolution of this disease. Gallic acid (3,4,5-trihydroxybenzoic acid), a common botanic phenolic compound, has been reported to induce apoptosis in tumor cell lines and renal fibroblasts. The present study was undertaken to examine the role of mitogen-activated protein kinases (MAPKs) in lung fibroblasts apoptosis induced by gallic acid. We found that treatment with gallic acid resulted in activation of c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and protein kinase B (PKB, Akt), but not p38MAPK, in mouse lung fibroblasts. Inhibition of JNK using pharmacologic inhibitor (SP600125) and genetic knockdown (JNK specific siRNA) significantly inhibited p53 accumulation, reduced PUMA and Fas expression, and abolished apoptosis induced by gallic acid. Moreover, treatment with antioxidants (vitamin C, N-acetyl cysteine, and catalase) effectively diminished gallic acid-induced hydrogen peroxide production, JNK and p53 activation, and cell death. These observations imply that gallic acid-mediated hydrogen peroxide formation acts as an initiator of JNK signaling pathways, leading to p53 activation and apoptosis in mouse lung fibroblasts.
Mitogen-activated protein kinases (MAPKs) are involved in the regulation of various cellular responses including cell proliferation, differentiation and survival. Although MAPKs are activated by MAPK kinase and inactivated by phosphatases, different types of MAPKs, including extracellular signal-regulated kinases (ERK1 and 2), c-jun N-terminal protein kinases (JNK) and p38 kinases are known to participate in different signalling pathways. This article will review some salient features of the regulation and function of different forms of MAPKs in the heart. Furthermore, the status of cardiac MAPKs under different pathophysiological conditions will be described.
A wide variety of external stimuli are known to activate MAPKs, which are then translocated from the cytoplasm to the nucleus and regulate cardiac gene expression by phosphorylating various transcriptional factors. By virtue of the involvement of ERK1/2 in hypertrophic response and of the stress-activated JNKs and p38 kinases in the process of apoptosis, MAPKs are considered to be intimately involved in cardiac remodelling. Both growth factors and phorbol esters have been shown to strongly activate ERK1/2, whereas the activation of JNKs and p38 kinases by these agents is weak. Although ischemia-reperfusion activates all types of MAPKs, JNKs and p38 kinases are mainly proapoptotic, whereas ERK1/2 are antiapoptotic.
The activation of ERK1/2 is involved in signal transduction pathways associated with cardiac hypertrophy; however, the exact status of MAPKs in heart failure remains to be clearly defined. While both JNKs and p38 kinases appear to participate in the genesis of ischemia-reperfusion injury, ERK1/2 are considered to be cytoprotective.
Cardiac hypertrophy; c-jun N-terminal protein kinases; Extracellular signal-regulated kinases; Heart failure; Mitogen-activated protein kinase; p38 kinases
Cellular phosphorylation events during viral infection are necessary for effective viral replication. Encephalomyocarditis (EMC) virus has been used for studies on the molecular mechanisms of viral replication, but little is known about the cellular signaling pathways involved. This investigation was initiated to determine whether mitogen-activated protein kinases (MAPKs), which are central components of signal transduction pathways in the regulation of cell proliferation, play a role in the replication of EMC virus. We examined the phosphorylation of MAPKs, including extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and stress-activated protein kinase 1/c-Jun NH2-terminal kinase (SAPK/JNK) in EMC virus-infected L929 cells and found that p38 MAPK and SAPK-JNK, but not ERK1/2, were activated during viral infection. We then examined the effect of these kinases on the replication of EMC virus in L929 cells by using specific inhibitors, including genistein or herbimycin A for tyrosine kinase, SB203580 or SB202190 for p38 MAPK, and PD98059 for ERK1/2. We found that the tyrosine kinase and p38 MAPK inhibitors, but not the ERK1/2 inhibitor, suppressed viral replication and that the inhibitory effect was primarily on viral protein synthesis. Finally, we examined whether p38 MAPK is involved in the translation of EMC viral transcripts by using L929 cells transfected with a gene construct containing the internal ribosomal entry site (IRES) of EMC virus and a luciferase reporter gene. We found that the p38 MAPK inhibitor suppressed the translation of EMC viral RNA. On the basis of these observations, we conclude that p38 MAPK plays a critical role in the replication of EMC virus, probably in the translation of viral RNA.
Vascular disrupting agents (VDAs), anti-cancer drugs that target established tumor blood vessels, fall into two main classes: microtubule targeting drugs, exemplified by combretastatin A4 (CA4), and flavonoids, exemplified by 5,6-dimethylxanthenone-4-acetic acid (DMXAA). Both classes increase permeability of tumor vasculature in mouse models, and DMXAA in particular can cause massive tumor necrosis. The molecular target of CA4 is clearly microtubules. The molecular target(s) of DMXAA remains unclear. It is thought to promote inflammatory signaling in leukocytes, and has been assumed to not target microtubules, though it is not clear from the literature how carefully this assumption has been tested. An earlier flavone analog, flavone acetic acid, was reported to promote mitotic arrest suggesting flavones might possess anti-microtubule activity, and endothelial cells are sensitive to even mild disruption of microtubules. We carefully investigated whether DMXAA directly affects the microtubule or actin cytoskeletons of endothelial cells by comparing effects of CA4 and DMXAA on human umbilical vein endothelial cells (HUVEC) using time-lapse imaging and assays for cytoskeleton integrity. CA4 caused retraction of the cell margin, mitotic arrest and microtubule depolymerization, while DMXAA, up to 500 µM, showed none of these effects. DMXAA also had no effect on pure tubulin nucleation and polymerization, unlike CA4. We conclude that DMXAA exhibits no direct anti-microtubule action and thus cleanly differs from CA4 in its mechanism of action at the molecular level.
Mitogen-activated protein kinase (MAPK) cascades control cell fate decisions, such as proliferation, differentiation, and apoptosis by integrating and processing intra- and extracellular cues. However, similar MAPK kinetic profiles can be associated with opposing cellular decisions depending on cell type, signal strength, and dynamics. This implies that signaling by each individual MAPK cascade has to be considered in the context of the entire MAPK network. Here, we develop a dynamic model of feedback and crosstalk for the three major MAPK cascades; extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38), c-Jun N-terminal kinase (JNK), and also include input from protein kinase B (AKT) signaling. Focusing on the bistable activation characteristics of the JNK pathway, this model explains how pathway crosstalk harmonizes different MAPK responses resulting in pivotal cell fate decisions. We show that JNK can switch from a transient to sustained activity due to multiple positive feedback loops. Once activated, positive feedback locks JNK in a highly active state and promotes cell death. The switch is modulated by the ERK, p38, and AKT pathways. ERK activation enhances the dual specificity phosphatase (DUSP) mediated dephosphorylation of JNK and shifts the threshold of the apoptotic switch to higher inputs. Activation of p38 restores the threshold by inhibiting ERK activity via the PP1 or PP2A phosphatases. Finally, AKT activation inhibits the JNK positive feedback, thus abrogating the apoptotic switch and allowing only proliferative signaling. Our model facilitates understanding of how cancerous deregulations disturb MAPK signal processing and provides explanations for certain drug resistances. We highlight a critical role of DUSP1 and DUSP2 expression patterns in facilitating the switching of JNK activity and show how oncogene induced ERK hyperactivity prevents the normal apoptotic switch explaining the failure of certain drugs to induce apoptosis.
dynamic model; bistability; JNK mitogen-activated protein kinases; Akt (PKB); dual specificity phosphatase
Fibroblast growth factor (FGF2), but not vascular endothelial growth factor (VEGF), upregulates endothelial nitric oxide synthase (eNOS) protein expression, at least in part, via activation of extracellular signal-regulated kinase 2/1 (ERK2/1) in ovine fetoplacental artery endothelial (oFPAE) cells. Herein we further investigated the temporal effects of FGF2 and VEGF on other signaling pathways including members (Jun N-terminal kinase JNK1/2 and p38MAPK) of mitogen-activated protein kinases (MAPK), phosphatidylinositol 3 kinase/v-akt murine thymoma viral oncogene homolog 1 (PI3K/AKT1), and the tyrosine kinase c-SRC, and examined if either one or more of these pathways play a role in the differential regulation of eNOS by FGF2 and VEGF. We first confirmed that in oFPAE cells, FGF2, but not VEGF, increased eNOS protein. FGF2 stimulated eNOS protein in a time and concentration dependent manner, which also depended on cell density. FGF2 provoked sustained (5 min to 12 h) whereas VEGF only stimulated transient (5 min) ERK2/1 phosphorylation. FGF2 was 1.7-fold more potent in stimulating ERK2/1 phosphorylation than VEGF. FGF2 and VEGF only transiently activated JNK1/2 and AKT1 within 5 min; however, FGF2 was a stronger stimulus than VEGF. FGF2 and VEGF did not significantly activate p38MAPK at 5 min; however, VEGF stimulated p38MAPK phosphorylation at 60 min. VEGF but not FGF2 significantly stimulated c-SRC phosphorylation. Inhibitors of MEK-ERK2/1 (PD98059), JNK1/2 (SP600125) and PI3K (wortmannin), but not p38MAPK (SB203580) and SRC (PP2), decreased the FGF2-increased eNOS protein expression. Thus, the FGF2-induced eNOS protein expression requires activation of multiple signaling pathways including ERK2/1, JNK1/2 and PI3K/AKT1. Differences in intensity and temporal patterns of activation of these pathways by FGF2 and VEGF may account for their differential effects on eNOS expression in OFPAE cells.
FGF2; VEGF; signaling pathways; eNOS protein; endothelial cells; placenta
In this report, we examined the antitumor activity of photodynamic therapy (PDT) in combination with 5,6-dimethylxanthenone- 4-acetic acid (DMXAA), a vascular disrupting agent currently undergoing clinical evaluation. BALB/c mice bearing subcutaneous CT-26 colon carcinomas were treated with PDT using the second-generation chlorin-based sensitizer, 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (Photochlor) with or without DMXAA. Long-term (60-days) treatment outcome, induction of tumor necrosis factor-alpha (TNF-α) and interleukin- 6 (IL-6), vascular damage (microvessel density, MVD) were evaluated as endpoints. In addition, treatment selectivity was evaluated using magnetic resonance imaging (MRI) and the foot response assay. A highly synergistic interaction was observed with the combination of low-dose DMXAA and PDT (48 J cm−2 at 112 mW cm−2) resulting in ~60% long-term cures. The duration of the PDT session for this combination therapy protocol was only 7 min, while the duration of a monotherapy PDT session, selected to yield the equivalent cure rate, was 152 min. MRI showed markedly less peritumoral edema after DMXAA + short-duration PDT compared with long-duration PDT monotherapy. Similarly, DMXAA + PDT caused significantly less phototoxicity to normal mouse foot tissue than PDT alone. Increased induction of cytokines TNF-α and IL-6 (P < 0.001) was observed at 4 h followed by extensive vascular damage, demonstrated by a significant reduction in MVD at 24 h after combination treatment. In conclusion, Photochlorsensitized PDT in combination with DMXAA exhibits superior efficacy and improved selectivity with clinically feasible illumination schemes. Clinical evaluation of this novel combination strategy is currently being planned.