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.
Vascular disrupting agents (VDAs) such as DMXAA (5,6-dimethylxanthenone-4-acetic acid) represent a novel approach for cancer treatment. DMXAA has potent anti-tumor activity in mice and, despite significant pre-clinical promise, failed human clinical trials. The anti-tumor activity of DMXAA has been linked to its ability to induce type I interferons in macrophages although the molecular mechanisms involved are poorly understood. Here we identify STING as a direct receptor for DMXAA leading to TBK1 and IRF3 signaling. Remarkably, the ability to sense DMXAA was restricted to murine STING. Human STING failed to bind to or signal in response to DMXAA. Human STING also failed to signal in response to cyclic-dinucleotides, conserved bacterial second messengers known to bind and activate murine STING signaling. Collectively, these findings detail an unexpected species-specific role for STING as a receptor for an anti-cancer drug and uncover important insights that may explain the failure of DMXAA in clinical trials for human cancer.
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.
The vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a murine agonist of the stimulator of interferon genes (STING), appears to target the tumor vasculature primarily as a result of stimulating pro-inflammatory cytokine production from tumor-associated macrophages (TAMs). Since there were relatively few reports of DMXAA effects in genetically-engineered mutant mice (GEMM), and models of non-small cell lung cancer (NSCLC) in particular, we examined both the effectiveness and macrophage dependence of DMXAA in various NSCLC models. The DMXAA responses of primary adenocarcinomas in K-rasLA1/+ transgenic mice, as well as syngeneic subcutaneous and metastatic tumors, generated by a p53R172HΔg/+; K-rasLA1/+ NSCLC line (344SQ-ELuc), were assessed both by in vivo bioluminescence imaging as well as by histopathology. Macrophage-dependence of DMXAA effects was explored by clodronate liposome-mediated TAM depletion. Furthermore, a comparison of the vascular structure between subcutaneous tumors and metastases was carried out using micro-computed tomography (micro-CT). Interestingly, in contrast to the characteristic hemorrhagic necrosis produced by DMXAA in 344SQ-ELuc subcutaneous tumors, this agent failed to cause hemorrhagic necrosis of either 344SQ-ELuc-derived metastases or autochthonous K-rasLA1/+ NSCLCs. In addition, we found that clodronate liposome-mediated depletion of TAMs in 344SQ-ELuc subcutaneous tumors led to non-hemorrhagic necrosis due to tumor feeding-vessel occlusion. Since NSCLC were comprised exclusively of TAMs with anti-inflammatory M2-like phenotype, the ability of DMXAA to re-educate M2-polarized macrophages was examined. Using various macrophage phenotypic markers, we found that the STING agonists, DMXAA and the non-canonical endogenous cyclic dinucleotide, 2′3′-cGAMP, were both capable of re-educating M2 cells towards an M1 phenotype. Our findings demonstrate that the choice of preclinical model and the anatomical site of a tumor can determine the vascular disrupting effectiveness of DMXAA, and they also support the idea of STING agonists having therapeutic utility as TAM repolarizing agents.
AIM: To explore the possibility that nucleotide oligomerization domain 1 (NOD1) pathway involved in refractoriness of interferon-β signaling in mouse respiratory epithelial cells induced by the anticancer xanthone compound, 5,6-dimethylxanthenone-4-acetic acid (DMXAA).
METHODS: C10 mouse bronchial epithelial cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 100 units/mL penicillin, 100 g/mL streptomycin. Pathogen-free female BALB/c mice were used to explore the mechanisms of refractoriness of interferon-signaling. Mouse thioglycollate-elicited peritoneal macrophages, bone marrow derived macrophages and bone marrow derived dendritic cells were collected and cultured. The amount of interferon (IFN)-inducible protein-10 (IP10/CXCL10), macrophage chemotactic protein (MCP1/CCL2) and interleukin (IL)-6 secreted by cells activated by DMXAA was quantified using enzyme-linked immunosorbent assay kits according to the instructions of the manufacturers. Total RNA was isolated from cells or nasal epithelium with RNeasy Plus Mini Kit, and cDNA was synthesized. Gene expression was checked using Applied Biosystems StepOne Real-Time Polymerase Chain Reaction System. Transfection of small interfering RNA (siRNA) control, NOD1 duplexed RNA oligonucleotides, and high-mobility group box 1/2/3 (HMGB1/2/3) siRNA was performed using siRNA transfection reagent.
RESULTS: DMXAA activates IFN-β pathway with high level of IFN-β dependent antiviral genes including 2’, 5’-oligoadenylate synthetase 1 and myxovirus resistance 1 in mouse thioglycollate-elicited peritoneal macrophages, bone marrow derived macrophages and bone marrow derived dendritic cells. Activation of IFN-β by DMXAA involved in NOD1, but not HMGB1/2/3 signal pathway demonstrated by siRNA. NOD1 pathway plays an important role in refractoriness of IFN-β signaling induced by DMXAA in mouse C10 respiratory epithelial cells and BALB/c mice nasal epithelia. These data indicate that DMXAA is not well adapted to the intrinsic properties of IFN-β signaling. Approaches to restore sensitivity of IFN-β signaling by find other xanthone compounds may function similarly, could enhance the efficacy of protection from influenza pneumonia and potentially in other respiratory viral infections.
CONCLUSION: NOD1 pathway may play an important role in refractoriness of IFN-β signaling in mouse respiratory epithelial cells induced by DMXAA.
Innate immunity; Interferon; Refractoriness; Xanthone; Bronchial epithelium
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.
Successful immunotherapy will require alteration of the tumour microenvironment and/or decreased immune suppression. Tumour-associated macrophages (TAMs) are one major factor affecting tumour microenvironment. We hypothesised that altering TAM phenotype would augment the efficacy of immunotherapy.
We and others have reported that 5,6-Dimethylxanthenone-4-acetic-acid (DMXAA, Vadimezan) has the ability to change TAM phenotypes, inducing a tumour microenvironment conducive to antitumour immune responses. We therefore combined DMXAA with active immunotherapies, and evaluated anti-tumour efficacy, immune cell phenotypes (flow cytometry), and tumour microenvironment (RT–PCR).
In several different murine models of immunotherapy for lung cancer, DMXAA-induced macrophage activation significantly augmented the therapeutic effects of immunotherapy. By increasing influx of neutrophils and anti-tumour (M1) macrophages to the tumour, DMXAA altered myeloid cell phenotypes, thus changing the intratumoural M2/non-M2 TAM immunoinhibitory ratio. It also altered the tumour microenvironment to be more pro-inflammatory. Modulating macrophages during immunotherapy resulted in increased numbers, activity, and antigen-specificity of intratumoural CD8+ T cells. Macrophage depletion reduced the effect of combining immunotherapy with macrophage activation, supporting the importance of TAMs in the combined effect.
Modulating intratumoural macrophages dramatically augmented the effect of immunotherapy. Our observations suggest that addition of agents that activate TAMs to immunotherapy should be considered in future trials.
tumour immunology; immunotherapy; tumour-associated macrophages; lung cancer
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.
Antigen-specific immunotherapy using DNA vaccines has emerged as an attractive approach for the control of tumors. Another novel cancer therapy involves the employment of the vascular disrupting agent, 5,6-dimethylxanthenone-4-acetic acid (DMXAA). In the current study, we aimed to test the combination of DMXAA treatment with human papillomavirus type 16 (HPV-16) E7 DNA vaccination to enhance the antitumor effects and E7-specific CD8+ T cell immune responses in treated mice. We determined that treatment with DMXAA generates significant therapeutic effects against TC-1 tumors but does not enhance the antigen-specific immune responses in tumor bearing mice. We then found that combination of DMXAA treatment with E7 DNA vaccination generates potent antitumor effects and E7-specific CD8+ T cell immune responses in the splenocytes of tumor bearing mice. Furthermore, the DMXAA-mediated enhancement or suppression of E7-specific CD8+ T cell immune responses generated by CRT/E7 DNA vaccination was found to be dependent on the time of administration of DMXAA and was also applicable to other antigen-specific vaccines. In addition, we determined that inducible nitric oxide synthase (iNOS) plays a role in the immune suppression caused by DMXAA administration before DNA vaccination. Our study has significant implications for future clinical translation.
Antigen-specific immunotherapy and vascular disrupting agents, such as 5,6-dimethylxanthenone-4-acetic acid (DMXAA), have emerged as attractive approaches for the treatment of cancers. In the current study, we tested the combination of DMXAA treatment with therapeutic human papillomavirus type 16 (HPV-16) E7 peptide-based vaccination for their ability to generate E7-specific CD8+ T-cell immune responses, as well as their ability to control E7-expressing tumors in a subcutaneous and a cervicovaginal tumor model. We found that the combination of DMXAA treatment with E7 long peptide (amino acids 43–62) vaccination mixed with polyriboinosinic:polyribocytidylic generated significantly stronger E7-specific CD8+ T-cell immune responses and antitumor effects compared with treatment with DMXAA alone or HPV peptide vaccination alone in the subcutaneous model. Additionally, we found that the DMXAA-mediated enhancement of E7-specific CD8+ T-cell immune responses generated by the therapeutic HPV peptide-based vaccine was dependent on the timing of administration of DMXAA. Treatment with DMXAA in tumor-bearing mice was also shown to lead to increased dendritic cell maturation and increased production of inflammatory cytokines in the tumor. Furthermore, we observed that the combination of DMXAA with HPV-16 E7 peptide vaccination generated a significant enhancement in the antitumor effects in the cervicovaginal TC-1 tumor growth model, which closely resembles the tumor microenvironment of cervical cancer. Taken together, our data demonstrated that administration of the vascular disrupting agent, DMXAA, enhances therapeutic HPV vaccine-induced cytotoxic T-lymphocyte responses and antitumor effects against E7-expressing tumors in two different locations. Our study has significant implications for future clinical translation.
Vascular disrupting agents such as 5,6-dimethylxanthenone-4-acetic acid (DMXAA) have emerged as a new class of potential anticancer drugs that selectively destroy the established tumor vasculature and shut down blood supply to solid tumors. In this study, Zeng and colleagues test the combination of DMXAA treatment with E7 long peptide vaccination mixed with poly(I:C) to enhance cellular immunity against E7-expressing tumors in mice.
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.
The investigative anti-tumour agent 5,6-dimethylxanthenonone-4-acetic acid (DMXAA, NSC 640488), developed in this laboratory as an improved analogue of flavone acetic acid (FAA, NSC 347512), is currently in clinical trial. The ability of DMXAA to up-regulate tumour necrosis factor (TNF) mRNA and protein synthesis in cultured human peripheral blood leucocytes (HPBLs) has been investigated and compared with that of flavone acetic acid (FAA) and of bacterial lipopolysaccharide (LPS). Human peripheral blood leucocytes were isolated from buffy coats obtained from a blood transfusion centre and also from blood samples from laboratory volunteers. At a concentration of 400 microg ml(-1) and an incubation time of 2 h, DMXAA up-regulated mRNA synthesis in six of eight individuals tested, as measured by Northern blotting. The degree of up-regulation varied in different individuals from one to nine times that of control levels. In contrast, FAA caused no induction above that of control levels and in some cases suppressed expression relative to controls, extending previous data that DMXAA but not FAA up-regulates TNF mRNA in the human HL-60 tumour cell line. At the same concentration but with longer incubation times (6-12 h), DMXAA induced increases in TNF protein in 11 of 15 samples of HPBLs from buffy coats and also in 11 of 15 samples of HPBLs from volunteers, as measured by cytotoxicity assays with L929 cells. FAA caused no increase in TNF protein, while LPS induced TNF to approximately 20-fold higher levels than did DMXAA. Considerable heterogeneity of response was observed with both sources of HPBLs, and there was little or no correlation between the extent of TNF induction by DMXAA and LPS in individual samples. In vitro analysis of the response of human peripheral blood leucocytes to DMXAA may be a useful test in clinical trials of agents such as DMXAA.
To investigate the early effects of a vascular disrupting agent (VDA) in ectopic and orthotopic tumors using macromolecular contrast media-enhanced magnetic resonance imaging (MMCM-MRI).
Methods and Materials
MMCM-MRI of ectopic and orthotopic MCA205 murine fibrosarcomas was performed using the intravascular contrast agent, albumin-(Gd-DTPA)35. Change in longitudinal relaxation rate (ΔR1) was measured 24 hours after treatment with 5,6-dimethylxanthenone-4-acetic acid (DMXAA; 30 mg/kg) and used to compute tumor vascular volume and permeability. Correlative histology and immunohistochemistry was carried out along with measurement of tumor necrosis factor-alpha (TNF-α) and vascular endothelial growth factor (VEGF) levels in whole tumor extracts using the enzyme-linked immunosorbent assay (ELISA).
Orthotopic tumors exhibited higher vascular volume (P<0.05) than ectopic tumors prior to treatment. Twenty-four hours after DMXAA treatment, a significant (P<0.0001) but differential reduction in ΔR1 (70% in ectopic and 50% in orthotopic tumors) was observed compared to baseline estimates. Consistent with this observation, higher levels of TNF-α, an important mediator of the antivascular activity of DMXAA were measured in ectopic tumors three hours post treatment compared to orthotopic tumors (P<0.05). Immunohistochemical (CD31) and histological (H&E) sections of ectopic and orthotopic tumors showed a highly tumor selective vascular damage following treatment with the presence of viable surrounding normal tissue.
MMCM-MRI provided early quantitative estimates of change in tumor perfusion following VDA treatment that showed good correlation with cytokine induction. Differences in the response of ectopic and orthotopic tumors highlight the influence of host microenvironment in modulating the activity of VDAs.
VDA; DMXAA; ASA404; MMCM-MRI
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 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.
The macrophage proinflammatory response to Francisella tularensis (Ft) LVS was shown previously to be TLR2-dependent. The observation that intracellular Ft LVS co-localizes with TLR2 and MyD88 inside macrophages suggested that Ft LVS might signal from within the phagosome. Macrophages infected with LVSΔiglC, a Ft LVS mutant that fails to escape from the phagosome, displayed greatly increased expression of a subset of TLR2-dependent, proinflammatory genes (e.g., Tnf) but decreased expression of others (e.g., Ifnb1). This latter subset was similarly mitigated in IFN-β−/− macrophages indicating that while Ft LVS-induced TLR2 signaling is necessary, cytosolic sensing of Ft to induce IFN-β is required for full induction of the macrophage proinflammatory response. While LVSΔiglC greatly increased IL-1β mRNA in wild-type macrophages, protein secretion was not observed. IL-1β secretion was also diminished in Ft LVS-infected IFN-β−/− macrophages. rIFN-β failed to restore IL-1β secretion in LVSΔiglC-infected macrophages, suggesting that signals in addition to IFN-β are required for assembly of the inflammasome and activation of caspase-1. IFN-β plays a central role in controlling the macrophage bacterial burden: bacterial recovery was greater in IFN-β−/− than in wild-type macrophages and treatment of Ft LVS-infected macrophages with rIFN-β or 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a potent IFN-β inducer, greatly decreased the intracellular Ft LVS burden. In toto, these observations support the hypothesis that the host inflammatory response to Ft LVS is complex and requires engagement of multiple signaling pathways downstream of TLR2 including production of IFN-β via an unknown cytosolic sensor and activation of the inflammasome.
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
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.
Hemagglutinin B (HagB) is a nonfimbrial adhesin expressed on the surface of Porphyromonas gingivalis and has been implicated as a potential virulence factor involved in mediating the attachment of the bacteria to host cells. However, the molecular mechanisms underlying host responses to HagB and their roles in pathogenesis have yet to be elucidated. Mitogen-activated protein kinases (MAPKs) are activated following engagement of a variety of cell surface receptors via dual tyrosine and threonine phosphorylation and are thought to be involved in various cellular responses. The purpose of this study was to determine the role of intracellular signaling pathways including the MAPKs and NF-κB in regulating the production of proinflammatory and anti-inflammatory cytokines following stimulation of murine macrophages with recombinant HagB (rHagB). Stimulation of peritoneal macrophages with rHagB resulted in the production of the proinflammatory cytokines interleukin-12p40 (IL-12p40), gamma interferon (IFN-γ), and tumor necrosis factor alpha, as well as the anti-inflammatory cytokine IL-10. We also demonstrated the activation of extracellular signal-related kinase (ERK), c-Jun NH2-terminal protein kinase (JNK), and p38 MAPKs by rHagB-stimulated macrophages. Furthermore, blocking of the ERK and p38 signaling pathways by using specific inhibitors revealed differential regulatory roles in the rHagB-mediated production of proinflammatory and anti-inflammatory cytokines. ERK and p38 were important in down-regulation of IL-12p40 and IFN-γ production and up-regulation of IL-10 production. The enhanced levels of IL-12p40 in rHagB-stimulated macrophages by inhibition of ERK or p38 activity were partially attributable to the inhibition of IL-10 production. Moreover, NF-κB was found to be critical for up-regulation of IL-12p40 and down-regulation of IL-10 production in rHagB-stimulated macrophages. Taken together, our results demonstrate a role for the p38 and ERK pathways and the transcription factor NF-κB in modulating key immunoregulatory cytokines involved in the development of immune responses to P. gingivalis HagB.
The flavonoids FAA and DMXAA showed impressive activity against solid tumors in mice, but failed clinical trials. They act on a previously unknown molecular target(s) to trigger cytokine release from leukocytes, which causes tumor-specific vascular damage and other anti-tumor effects. We show that DMXAA is a competitive agonist ligand for mouse STING (stimulator of interferon genes), a receptor for the bacterial PAMP cyclic-di-GMP (c-di-GMP) and an endogenous second messenger cyclic-GMP-AMP. In our structure-activity relationship studies, STING binding affinity and pathway activation activity of four flavonoids correlated with activity in a mouse tumor model measured previously. We propose that STING agonist activity accounts for the anti-tumor effects of FAA and DMXAA in mice. Importantly, DMXAA does not bind to human STING, which may account for its lack of efficacy or mechanism-related toxicity in man. We propose that STING is a druggable target for a novel innate immune activation mechanism of chemotherapy.
The sustenance of most solid tumors including head and neck cancers (HNCs) is strongly dependent on the presence of a functioning vascular network. In this study, we examined the acute effects of a tumor vascular disrupting agent (VDA), 5,6-dimethylxanthenone-4-acetic acid (DMXAA; ASA404), in an orthotopic model of human HNC. Noninvasive magnetic resonance imaging (MRI) was used to monitor the vascular response of orthotopic FaDu xenografts to VDA therapy. Untreated tumors showed a marked but heterogeneous pattern of enhancement after contrast agent injection on serial T1-weighted (T1W) MR images. After VDA treatment, T2W and T1W MRI revealed evidence of hemorrhaging and lack of functioning vessels (enhancement) within the tumor. Quantitative estimates of relative vascular volume also showed a significant (P < .01) reduction in DMXAA-treated tumors 24 hours after therapy compared with untreated controls. Histology and immunostaining of untreated orthotopic FaDu tumors revealed poorly differentiated squamous cell carcinoma histology with distinctly visible CD31+ endothelial cells. In sharp contrast, minimal CD31 staining with irregular endothelial fragments and faint outlines of blood vessels were seen in DMXAA-treated tumor sections. CD31 immunostaining and histology also highlighted the selectivity of vascular damage and tissue necrosis after VDA therapy with no evidence of toxicity observed in normal salivary gland, heart, liver, and skeletal muscle tissues. Together, our results demonstrate a potent and selective vascular disruptive activity of DMXAA in an orthotopic HNC model. Further evaluation into its antitumor effects alone and in combination with other agents is warranted.
Head and neck squamous cell carcinomas (HNSCC) constitute a majority of the tumors of the upper aerodigestive tract and continue to present a significant therapeutic challenge. To explore the potential of vascular-targeted therapy in HNSCC, we investigated the antivascular, antitumor activity of the potent vascular-disrupting agent (VDA) 5,6-dimethylxanthenone-4-acetic acid (DMXAA) against two HNSCC xenografts with markedly different morphologic and vascular characteristics. Athymic nude mice bearing subcutaneous FaDu (human pharyngeal squamous cell carcinoma) and A253 (human submaxillary gland epidermoid carcinoma) tumors were administered a single dose of DMXAA (30 mg/kg, i.p). Changes in vascular function were evaluated 24 hours after treatment using contrast-enhanced magnetic resonance imaging (MRI) and immunohistochemistry (CD31). Signal enhancement (E) and change in longitudinal relaxation rates (ΔR1) were calculated to measure alterations in vascular perfusion. MRI showed a 78% and 49% reduction in vascular perfusion in FaDu and A253 xenografts, respectively. CD31-immunostaining of tumor sections revealed three-fold (FaDu) and two-fold (A253) reductions in microvessel density (MVD) 24 hours after treatment. DMXAA was equally effective against both xenografts, with significant tumor growth inhibition observed 30 days after treatment. These results indicate that DMXAA may be beneficial in the management of HNSCC, alone or in combination with other treatments.
Head and neck cancers; DMXAA; tumor vasculature; MRI; antivascular therapies
Epigallocatechin-3-gallate (EGCG) is a bioactive polyphenol of green tea and exerts potent anti-inflammatory effects by inhibiting signaling events and gene expression. Interleukin-1beta (IL-1β) is the principal cytokine linked to cartilage degradation in osteoarthritis (OA). The objective of this study was to evaluate the global effect of EGCG on IL-1β-induced expression of proteins associated with OA pathogenesis in human chondrocytes.
Primary OA chondrocytes were pretreated with EGCG (10 to 100 uM) and then stimulated with IL-1β (5 ng/ml) for 24 hours. Culture supernatants were incubated with cytokine antibody arrays and immunoreactive proteins (80 proteins) were visualized by enhanced chemiluminiscence. Effect of EGCG on IL-1β-induced expression of 18 selected genes was verified by Real time-PCR and effect on IL-6, IL-8 and tumor necrosis factor-alpha (TNF-α) production was determined using specific ELISAs. Western immunoblotting was used to analyze the effect of EGCG on the interleukin-1 receptor-associated kinase 1 (IRAK-1) and TNF receptor-associated factor 6 (TRAF-6) proteins in IL-1β-stimulated chondrocytes. The role of nuclear factor kappa-B (NF-κB) and mitogen activated protein kinases (MAPKs) in the regulation of selected genes and the mechanism involved in EGCG mediated modulation of these genes was determined by using specific inhibitors for NF- κB (MG132) and MAPKs (p38-MAPK, SB202190; JNK-MAPK, SP600125, ERK-MAPK, PD98059).
Out of 80 proteins present on the array, constitutive expression of 14% proteins was altered by EGCG treatment. No significant stimulatory effect was observed on the proteins associated with cartilage anabolic response. Stimulation with IL-1β enhanced the expression of 29 proteins. Expression of all 29 proteins up-regulated by IL-1β was found to be suppressed by EGCG. EGCG also inhibited the expression of the signaling intermediate TRAF-6 at 50 and 100 uM concentrations (P < 0.05). Our results identified several new targets of EGCG, including epithelial neutrophil activating peptide-78 (ENA-78), granulocyte macrophage colony stimulation factor (GM-CSF), growth- related oncogene (GRO), GRO-α, IL-6, IL-8, monocyte chemotactic protein-1 (MCP-1), MCP-3, macrophage inflammatory protein-1beta (MIP-1β), granulocyte chemotactic protein-2 (GCP-2), MIP-3alpha, interferon-gamma-inducible protein-10 (IP-10), nucleosome assembly protein-2 (NAP-2) and leukemia inhibitory factor (LIF). The inhibitory effects of EGCG were mainly mediated by inhibiting the activation of NF-κB and c-Jun N-terminal Kinase (JNK)-MAPK in human chondrocytes.
Our results suggest that the potential of EGCG in OA treatment/prevention may be related to its ability to globally suppress the inflammatory response in human chondrocytes. These results identify additional new targets of EGCG and advocate that EGCG may be a potent chondroprotective agent in OA.