The use of tissue inhibitors of matrix metalloproteinases to increase the efficacy of a tumor necrosis factor/interferon gamma antitumor therapy

Owing to its impressive ability to kill tumor cells, especially in combination with interferon-gamma (IFNgamma), tumor necrosis factor (TNF) is widely appreciated as being a potential systemic therapeutic for the treatment of cancer. On the other hand, owing to its proinflammatory activities, administration of TNF leads to many systemic side effects and eventually to a potentially lethal systemic inflammatory response syndrome (SIRS). However, systemic treatment of tumor-bearing mice with TNF/IFNgamma in combination with BB-94 (a broad-spectrum metalloproteinase inhibitor) confers protection against TNF/IFNgamma-induced mortality, whereas preserving the antitumor activity. In this study, we investigated the effect of the adenoviral delivery of human tissue inhibitors of matrix metalloproteinase (hTIMP)-1 and hTIMP-2 genes on the outcome of TNF/IFNgamma antitumor therapy. The dose of adenovirus was limited to 10(8) PFU per mouse owing to the additive toxicity of combining it with TNF/IFNgamma therapy. Nevertheless, this dose was sufficient to achieve highly efficient adenoviral transfer and expression of hTIMP-1 and hTIMP-2 in the liver, but not the tumor. Treatment with this low dose of AdhTIMP-1 or AdhTIMP-2 was not enough to protect the host against the toxic effects of TNF/IFNgamma. However, it was sufficient to show a synergistic effect of hTIMPs with TNF/IFNgamma such that tumors regressed significantly faster. Interestingly, only AdTIMP-2 was able to prevent relapses after treatment.     

Insulin-like growth factor-I protects colon cancer cells from death factor-induced apoptosis by potentiating tumor necrosis factor alpha-induced mitogen-activated protein kinase and nuclear factor kappaB signaling pathways

Resistance of cancer cells against apoptosis induced by death factors contributes to the limited efficiency of immune- and drug-induced destruction of tumors. We report here that insulin and insulin-like growth factor-I (IGF-I) fully protect HT29-D4 colon carcinoma cells from IFN-gamma/tumor necrosis factor-alpha (TNF) induced apoptosis. Survival signaling initiated by IGF-I was not dependent on the canonical survival pathway involving phosphatidylinositol 3'-kinase. In addition, neither pp70(S6K) nor protein kinase C conveyed IGF-I antiapoptotic function. Inhibition of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) with the MAPK/ERK kinase inhibitor PD098059 and MAPK/p38 with the specific inhibitor SB203580 partially reversed, in a nonadditive manner, the IGF-I survival effect. Inhibition of nuclear factor kappaB (NF-kappaB) activity by preventing degradation of the inhibitor of NF-kappaB (IkappaB-alpha) with BAY 11-7082 also blocked in part the IGF-I antiapoptotic effect. However, the complete reversal of the IGF-I effect was obtained only when NF-kappaB and either MAPK/ERK or MAPK/p38 were inhibited together. Because these pathways are also those used by TNF to signal inflammation and survival, these data point to a cross talk between IGF-I- and TNF-induced signaling. We further report that TNF-induced IL-8 production was indeed strongly enhanced upon IGF-I addition, and this effect was totally abrogated by both MAPK and NF-kappaB inhibitors. The IGF-I antiapoptotic function was stimulus-dependent because Fas- and IFN/Fas-induced apoptosis was not efficiently inhibited by IGF-I. This was correlated with the weak ability of Fas ligation to enhance IL-8 production in the presence or absence of IGF-I. These findings indicate that the antiapoptotic function of IGF-I in HT29-D4 cells is based on the enhancement of the survival pathways initiated by TNF, but not Fas, and mediated by MAPK/p38, MAPK/ERK, and NF-kappaB, which act in concert to suppress the proapoptotic signals. In agreement with this model, we show that it was possible to render HT29-D4 cells resistant to Fas-induced apoptosis provided that IGF-I and TNF receptors were activated simultaneously.     

Interleukin 1, interleukin 6, tumor necrosis factor, and transforming growth factor beta increase cell resistance to tumor necrosis factor cytotoxicity by growth arrest in the G1 phase of the cell cycle

During infection, inflammation, immune responses, and neoplastic growth, various cytokines are produced affecting both susceptibility to and protection from cellular death. We have studied the protective effect of pretreatment of the L929 fibroblast cell line with interleukin 1 beta (IL-1 beta), IL-6, tumor necrosis factor alpha (TNF-alpha), or transforming growth factor beta 1 (TGF-beta) on subsequent TNF/actinomycin D-induced cytotoxicity. The protective effects of these cytokines on TNF cytotoxicity were time and concentration dependent. TGF-beta was the most effective cytokine, followed by TNF, IL-1 beta, and IL-6. Activators of protein kinase C also afforded protection, and TGF-beta acted synergistically with either phorbol 12-myristate 13-acetate or the calcium ionophore A-23187. TGF-beta-induced protection against TNF was observed in cells subjected to prolonged treatment with phorbol 12-myristate 13-acetate. Cells pretreated with prostaglandin E2 or cholera toxin amplified the sensitivity to TNF and inhibited TGF-beta-mediated resistance, whereas indomethacin enhanced the protective effect of TGF-beta. Cells cultured in the presence of IL-1 beta, IL-6, TNF-alpha, or TGF-beta for 6 h inhibited DNA synthesis, and this was associated with concomitant growth arrest in the G1 phase of the cell cycle. On the other hand, prostaglandin E2 or cholera toxin stimulated the progression of cells from G1 toward G2 + M which was associated with increased TNF sensitivity. We conclude that these cytokines protect against death by arresting growth in the G1 phase of the cell cycle.     

Regulation of components of the inflammatory response by 5-iodo-6-amino-1,2-benzopyrone, an inhibitor of poly(ADP-ribose) synthetase and pleiotropic modifier of cellular signal pathways

Chronic inflammation is known to facilitate carcinogenic transformation in various tissues. 5-iodo-6-amino-1,2-benzopyrone (INH2BP), a novel inhibitor of the nuclear enzyme poly(ADP-ribose) polymerase (pADPRT) has recently been shown to regulate a variety of cellular signal transduction pathways and to abrogate in vivo tumorigenicity by a Ha-ms transfected endothelial cell line. Here we have investigated the effect of INH2BP on the activation by endotoxin (bacterial lipopolysaccharide, LPS) on the production of the inflammatory mediators tumor necrosis factor alpha (TNF alpha), interleukin-10 (IL-10) and interleukin-6 (IL-6), nitric oxide (NO) and prostaglandins in vitro and in vivo. In addition, we studied the effect of INH2BP on the activation of mitogen-activated protein kinase (MAP kinase) and nuclear factor kappa B (NF-kappa B) in vitro. In cultured J774 and RAW 264.7 macrophages, LPS induced the production of prostaglandin metabolites, the release of TNF and the expression of the inducible isoform of NO synthase (iNOS). The production of prostaglandins and of NO were inhibited by INH2BP in a dose-dependent manner, while the short-term release of TNF alpha was unaffected. INH2BP markedly suppressed LPS-mediated luciferase activity in RAW cells transiently transfected with a full length (-1,592 bp) murine macrophage iNOS promoter-luciferase construct, but not in a deletional construct consisting of -367 bp. In vivo, INH2BP pretreatment inhibited the induction of iNOS by LPS in rats, did not affect the LPS-induced TNF and IL-6 response, but enhanced LPS-induced IL-10 production. INH2BP pretreatment markedly improved the survival of mice in a lethal model of endotoxin shock. Our results demonstrate that INH2BP has potent anti-inflammatory actions in vitro and in vivo.     

ONO-4007, a synthetic lipid A analog,induces Th1-type immune response in tumor eradication and restores nitric oxide production by peritoneal macrophages

We have reported that ONO-4007, a novel synthetic lipid A derivative with low toxic activities, produced large amount of tumor necrosis factor (TNF)-alpha selectively in the tumor tissues and brought about complete cures in about 60% of rats bearing TNF-alpha sensitive KDH-8 cells, which also secreted a large amount of transforming growth factor (TGF)-beta. In our present study, to explore ONO-4007 induced Th1-type immune response, we investigated the mRNA expression of interferon (IFN)-gamma, Interleukin (IL)-1beta, IL-6, IL-10, IL-12 in KDH-8 bearing rats. We next examined the nitric oxide (NO) production. We found that IFN-gamma, IL-1beta, IL-6 and IL-12 mRNA expression of the tumor tissue were higher in the ONO-4007 treated rats than in phosphate buffer saline (PBS) treated rats. Western blotting also revealed that IL-12 protein production was increased. NO production from peritoneal macrophages were suppressed in tumor-bearing rats, but ONO-4007 restored it up to the normal level. These results suggest that ONO-4007 induces and restores Th1-type immune response through cytokine production cascade, followed by initial TNF-alpha production, eventually leading to tumor eradication.     

TNF expressed by tumor-associated macrophages, but not microglia, can eliminate glioma

It is well known that tumor necrosis factor (TNF) can have both contrary and pleiotropic effects in anti-tumor immune response. In the present study, we prepared two different tumor cell-based immunotherapy models: MCA38 adenocarcinoma and GL261 glioma intracranial interleukin-2 (IL-2)-based. Each tumor was transfected to express IL-2 with or without expression of the soluble form of tumor necrosis factor receptor type II (sTNFRII). Although mice in which TNF is blocked survive longer than IL-2 alone (35.2 versus 26 days), the reverse was observed for GL261 glioma. The differential effect on tumor growth implies enhanced TNF sensitivity of GL261 compared to MCA38. This notion is supported by the observation that TNF induces apoptosis in GL261 but not MCA38 tumors. We further examined tumor infiltrating CD11b+F4/80+ macrophages (or tumor-associated macrophages: TAM) for TNF production in vivo and found that TAM express cell surface TNF implying a role in eliminating glioma cells mediated by the cell surface form of TNF.     

Integrin-linked kinase activity is associated with interleukin-1 alpha-induced progressive behavior of pancreatic cancer and poor patient survival

Cancer cell adhesion and invasion into extracellular matrix are regulated by integrin-linked kinase (ILK) activity in a phosphatidylinositol 3-kinase (PI3-K)-dependent manner. In this study, we demonstrated that ILK and beta(1)-integrin play important roles in interleukin (IL)-1alpha-induced enhancement of adhesion and invasion of pancreatic cancer cells through p38 mitogen-activated protein kinase (MAPK) signaling pathway and activator protein-1 (AP-1) activation. Alteration of ILK kinase activity controlled IL-1alpha-induced p38 MAPK phosphorylation and its downstream AP-1 activation with subsequent regulation of pancreatic cancer cell adhesion and invasion. Overexpressed ILK enhances the IL-1alpha-induced p38 MAPK phosphorylation more strongly through glycogen synthase kinase 3 (GSK-3) activation, and subsequently induces AP-1 activation, which promotes aggressive capabilities of pancreatic cancer cells. In contrast, knockdown of ILK kinase activity inhibits the IL-1alpha-induced activation of MAPK/AP-1 pathway via inhibition of GSK-3 phosphorylation. In immunohistochemical analysis, statistically significant association between strong expression of ILK and poor prognosis of pancreatic cancer patients were observed, and strong expression of ILK in cancerous tissues can be a significant prognostic indicator of pancreatic cancer patients. Our results suggest that ILK is involved with aggressive capability in pancreatic cancer and that these regulations can be helpful to understand biological processes for a better translational treatment for pancreatic cancer patients.     

Functional gene expression profile underlying methotrexate-induced senescence in human colon cancer cells

Cellular functions accompanying establishment of premature senescence in methotrexate-treated human colon cancer C85 cells are deciphered in the present study from validated competitive expression microarray data, analyzed with the use of Ingenuity Pathways Analysis (IPA) software. The nitrosative/oxidative stress, inferred from upregulated expression of inducible nitric oxide synthase (iNOS) and mitochondrial dysfunction-associated genes, including monoamine oxidases MAOA and MAOB, β-amyloid precursor protein (APP) and presenilin 1 (PSEN1), is identified as the main determinant of signaling pathways operating during senescence establishment. Activation of p53-signaling pathway is found associated with both apoptotic and autophagic components contributing to this process. Activation of nuclear factor κB (NF-κB), resulting from interferon γ (IFNγ), integrin, interleukin 1β (IL-1β), IL-4, IL-13, IL-22, Toll-like receptors (TLRs) 1, 2 and 3, growth factors and tumor necrosis factor (TNF) superfamily members signaling, is found to underpin inflammatory properties of senescent C85 cells. Upregulation of p21-activated kinases (PAK2 and PAK6), several Rho molecules and myosin regulatory light chains MYL12A and MYL12B, indicates acquisition of motility by those cells. Mitogen-activated protein kinase p38 MAPK β, extracellular signal-regulated kinases ERK2 and ERK5, protein kinase B AKT1, as well as calcium, are identified as factors coordinating signaling pathways in senescent C85 cells.     

Cytokine-suppressive anti-inflammatory drugs (CSAIDs) inhibit invasion and MMP-1 production of ovarian carcinoma cells

The development of therapeutic strategies for inhibition of peritoneal dissemination and invasion would be central for the treatment of ovarian carcinoma. In the microenvironment of ovarian carcinomas, various inflammatory cytokines like tumor necrosis factor alpha (TNF-alpha) are present. In this study we investigated the role of inflammatory cytokines in the regulation of invasion of SKOV-3 ovarian carcinoma cells in-vitro. Treatment of cells with TNF-alpha or interleukin 1beta (IL-1beta) lead to increased phosphorylation of the stress-activated p38 mitogen-activated protein kinase (p38MAPK). Furthermore, TNF-alpha as well as IL-1beta stimulated matrigel invasion of tumor cells. An inhibitor of stress-activated protein kinase pathways, the cytokine-suppressive anti-inflammatory drug (CSAID) SB203580 inhibited invasion of cytokine-stimulated SKOV-3 cells. The MEK-1 inhibitor PD98059 similarly inhibited invasion of cytokine-stimulated cells, but to a lesser extent. Expression of mRNA and protein levels of matrix metalloproteinase-1 (MMP-1) by SKOV-3 cells could be stimulated by inflammatory cytokines and inhibited by SB203580, and partially also by PD98059. Our results show that CSAIDs reduce invasion and MMP expression of ovarian carcinoma cells. Further studies are required to investigate whether inhibition of cytokine-induced signal transduction may be of value in therapy of ovarian carcinomas in-vivo.     

IL-4 regulation of IL-6 production involves Rac/Cdc42- and p38 MAPK-dependent pathways in keratinocytes

The stress-activated pathways leading to activation of p38 MAP kinase (p38 MAPK) and c-jun N-terminal kinases (JNK) have been shown to be activated by pro-inflammatory cytokines, physical and chemical stresses as well as a variety of hematopoietic growth factors. One exception is interleukin (IL)-4, which does not activate this pathway in hematopoietic cell. We report here that in A431, a keratinocytic cell line, IL-4 activates Rac and Cdc42 and their downstream effector p21-activated kinase (PAK). Rac and Cdc42 appear to regulate a protein kinase cascade initiated at the level of PAK and leading to activation of p38 MAPK, since IL-4 stimulates tyrosine phosphorylation of p38 MAPK and increases its catalytic activity. As A431 cells are able to produce IL-6 in response to IL-4 stimulation, we assessed the involvement of p38 MAPK in IL-6 gene expression. A pyrimidazole compound, SB203580, a specific inhibitor of p38 MAPK, inhibits production and gene expression of IL-6. SB203580 reduced significantly the stability of IL-6 mRNA. Here we provide evidence that p38 MAPK is activated in response to IL-4 and is involved in IL-6 synthesis by stabilizing IL-6 mRNA.     

Oncogenic Ras modulates p38 MAPK-mediated inflammatory cytokine production in glioblastoma cells

Inflammation is an important factor promoting the progression of glioblastoma. In the present study we examined the contribution of Ras signaling and TNFα/IL-1β cytokines to the development of the glioblastoma inflammatory microenvironment. Enhanced activation of Ras through de-regulated activation of receptor tyrosine kinases, such as EGFR, PDGFR and cMet, is a hallmark of the majority of glioblastomas. Glioblastoma microenvironment contains high levels of TNFα and IL-1β, which mediate inflammation through induction of a local network of cytokines and chemokines. While many studies have focused on Ras- and TNFα/IL-1β-driven inflammation in isolation, little is known about the co-operation between these oncogenic and microenvironment-derived stimuli. Using constitutively active HRasG12V that mimics enhanced Ras activation, we demonstrate that elevated Ras activity in glioblastoma cells leads to up-regulation of IL-6 and IL-8. Furthermore, Ras synergizes with the microenvironment-derived TNFα and IL-1β resulting in amplified IL-6/IL-8 secretion. IL-8 secretion induced by Ras and TNFα/IL-1β is attenuated by inhibitors targeting Erk, JNK and p38 MAPK pathways. IL-6 secretion significantly decreased upon inhibition of JNK and p38 MAPK pathways. Interestingly, although constitutively active HRasG12V does not increase basal or TNFα/IL-1β stimulated p38 MAPK activity, HRasG12V increased the efficacy of the p38 MAPK inhibitor SB203580 to inhibit IL-1β-induced IL-6 secretion. In summary, oncogenic Ras co-operates with the microenvironment-derived TNFα/IL-1β to sustain inflammatory microenvironment, which was effectively attenuated via inhibition of p38 MAPK signaling.     

Tumoricidal activity of high-dose tumor necrosis factor-alpha is mediated by macrophage-derived nitric oxide burst and permanent blood flow shutdown

This study investigates the role of tumor nitric oxide (NO) and vascular regulation in tumor ulceration following high-dose tumor necrosis factor-alpha (TNF) treatment. Using TNF-responsive (MethA) and nonresponsive (LL2) mouse tumors, tumor NO concentration was measured with an electrochemical sensor and tumor blood flow by Doppler ultrasound. Mice were also pretreated with a selective inducible nitric oxide synthase (iNOS) inhibitor, 1400 W. Tumors harvested from TNF-treated mice were cryosectioned and immunostained for murine macrophages, or/and iNOS. MethA tumor-bearing mice were depleted of macrophages. Pre- and post-TNF tumor NO levels were measured continuously, and mice were followed for gross tumor response. In MethA tumors, TNF caused a 96% response rate, and tumor NO concentration doubled. Tumor blood flow decreased to 3% of baseline by 4 hr and was sustained at 24 hr and 10 days post-TNF. Selective NO inhibition with 1400 W blocked NO rise and decreased response rate to 38%. MethA tumors showed tumor infiltration by macrophages post-TNF and the pattern of macrophage immunostaining overlapped with iNOS immunostaining. Depletion of macrophages inhibited tumor NO increase and response to TNF. LL2 tumors had a 0% response rate to TNF and exhibited no change in NO concentration. Blood flow decreased to 2% of baseline by 4 hr, recovered to 56% by 24 hr and increased to 232% by 10 days. LL2 tumors showed no infiltration by macrophages post-TNF. We conclude that TNF causes tumor infiltrating, macrophage-derived iNOS-mediated tumor NO rise and sustained tumor blood flow shutdown, resulting in tumor ulceration in the responsive tumor.     

Nitric oxide synthase is induced in tumor promoter-sensitive, but not tumor promoter-resistant, JB6 mouse epidermal cells cocultured with interferon-gamma-stimulated RAW 264.7 cells: the role of tumor necrosis factor-alpha

Expression of inducible nitric oxide synthase (iNOS) has been reported to be involved in certain organs of potential tumorigenesis, including the stomach and colon. The mechanisms for iNOS expression in epithelial cells, however, are not fully understood. In the present study, we investigated the role of macrophages in epithelial iNOS expression by coculturing a stimulated murine macrophage-like cell line, RAW 264.7, with either tumor promoter-sensitive (P+) or promoter-resistant (P-) JB6 murine epidermal cells. After monoculture, treatment of RAW 264.7 cells with IFN-gamma for 24 h generated a large amount of nitrite (NO2-), as reported previously, whereas no increase in NO2- concentration was observed in the IFN-gamma-treated P+ or P-subclones. Interestingly, when IFN-gamma-treated RAW 264.7 cells were cocultured with P+ but not P- cells, we observed a marked increase in NO2- concentration (30.8+/-3.6 microM), which significantly exceeded (P < 0.01) the sum of the concentrations (20.0+/-2.3 microM) added from each cell line monoculture. Western blotting analysis revealed that, after coculture, iNOS protein was up-regulated 55-fold more than the control in JB6 P+ but not in P- cells. IFN-gamma-treated RAW 264.7 cells secreted proinflammatory cytokines, including tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta. The addition of IFN-gamma-treated RAW 264.7 cell-conditioned media to P+ subclones led to a significant enhancement of NO2- formation that was diminished by the TNF-alpha-specific but not IL-1beta-specific antibody. When combined with IFN-gamma, the recombinant TNF-alpha (1-100 ng/ml) enhanced NO2- formation in JB6 P+ cells, whereas IL-1beta (1-100 ng/ml) did not. These results led us to conclude that IFN-gamma-treated RAW 264.7 cells release TNF-alpha to induce iNOS expression in promoter-sensitive JB6 cells. Thus, we propose the hypothesis that macrophages stimulate neoplastic cells with TNF-alpha via a paracrine loop to induce epithelial iNOS protein expression.     

Activation of p38 MAPK and/or JNK contributes to increased levels of VEGF secretion in human malignant glioma cells

Malignant gliomas are typically angiogenic and secrete high levels of VEGF. Hypoxia has been identified as an important regulator of VEGF. However, malignant gliomas express high levels of VEGF in both hypoxic perinecrotic and vital tumor areas. In this study, we examined intracellular signaling pathways involved in the secretion of VEGF in glioma cells under normoxic conditions. Human malignant glioma cell lines, T98G, U373MG, U87MG, and A172, and human fetal lung fibroblasts (HFL) were cultured both with and without IL-1beta under normoxic conditions. VEGF, IL-1, IL-6, and TNF-alpha were measured with ELISA. VEGF mRNA levels were estimated by RT-PCR. Inhibitors of COX-2, MAPK, and phosphatidyl inositol 3-kinase (PI3-K), and blocking antibodies to TGF-beta II and TNF-alpha, or IL-1 receptor antagonist, were used to examine their effects on VEGF secretion. Phosphorylation of MAPK was examined by immunoblotting. The basal levels of VEGF secretion were significantly higher in U87MG, U373MG, and T98G, than HFL. IL-1beta significantly stimulated VEGF secretion in these glioma cells. Inhibitors of p38 MAPK and/or JNK significantly suppressed VEGF secretion both in the presence and absence of IL-1beta, while inhibitors of COX-2, ERK1/2, and PI3-K did not. Constitutive phosphorylation of p38 MAPK and JNK was observed in these glioma cells. The levels of IL-1beta in U87MG were significantly higher than in other glioma cell lines, and IL-1 receptor antagonist suppressed basal secretion of VEGF from U87MG. In conclusion, p38 MAPK and JNK pathways play an important role in VEGF secretion from malignant glioma cells under normoxic conditions, possibly contributing to VEGF-induced angiogenesis in malignant gliomas at vital tumor areas where there is no hypoxia.     

EFFECTS OF PHYTOLACCA ACINOSA POLYSACCHARIDES I ON CYTOTOXICITY OF MACROPHAGES AND ITS PRODUCTION OF TUMOR NECROSIS FACTOR AND INTERLEUKIN 1

The in vivo effects of Phytolacca acinosa poly-saccharides I (PEP-I) on immunologic cytotoxicity of mouse peritoneal macrophages and its production of tumor necrosis factor (TNF) and interleukin 1 (IL-1) were studied. PEP-I 80 or 160 mg kg was given ip twice every 4 day. Both doses were found to have significant enhancing activity on macrophages cytotoxicity against S180 sarcoma cells and malignant transformed fibroblast L929 cells. Peritoneal activated macrophages were incubated with LPS for 2 and 24 hrs to induce TNF and IL-1, respectively. The TNF and IL-1 activities were tested from cytotoxicity against L929 cells in an absorbence assay of enzymatic reaction and proliferation of thymocytes co-stimulated assay separately. The optimal time for TNF production was found on day 8. Significant increases in TNF and IL-1 were observed. In comparison of the effect of PEP-I on TNF with that of known priming agent BCG, there was no difference between them, but PEP-I had a high effect on IL-1. These results sug