Glyceryl trinitrate­induced cytotoxicity of docetaxel­resistant prostatic cancer cells is associated with differential regulation of clusterin.

Metastatic castration resistant prostate cancer (mCRPC) relapse due to acquired resistance to chemotherapy, such as docetaxel, remains a major threat to patient survival. Resistance of mCRPC to docetaxel can be associated with elevated levels of soluble clusterin (sCLU) and growth differentiation factor­15 (GDF­15). Any strategies aiming to modulate sCLU and/or GDF­15 in docetaxel­resistant prostate cancer cells present a therapeutic interest. The present study reports the cytotoxic effect of a nitric oxide donor, glyceryl trinitrate (GTN), on docetaxel­resistant mCRPC human cell lines and demonstrates that GTN displays greater inhibition of cell viability toward docetaxel­resistant mCRPC cells than on mCRPC cells. It is also demonstrated that GTN modulates the level of expression of clusterin (CLU) which is dependent of GDF­15, two markers associated with docetaxel resistance in prostate cancer. The results indicate that GTN represses the level of expression of the cytoprotective isoform of CLU (sCLU) and can increase the level of expression of the cytotoxic isoform (nuclear CLU) in docetaxel resistant cells. Furthermore, it was observed that GTN differentially regulates the level of the precursor form of GDF­15 between resistant and parental cells, and that recombinant GDF­15 can modulate the expression of CLU isoforms and counteract GTN­induced cytotoxicity in resistant cells. A link was established between GDF­15 and the expression of CLU isoforms. The present study thus revealed GTN as a potential therapeutic strategy to overcome docetaxel­resistant mCRPC.

Tumor-derived granzyme B-expressing neutrophils acquire antitumor potential after lipid A treatment.

Neutrophils are known to possess both pro- and anti-tumor properties, a feature that could be related to the diversity and plasticity of these cells. Here we explored the hypothesis that under an appropriate environment and stimuli, neutrophils could induce an effective response against tumor cells. In a rat and mouse models, we show that a substantial amount of colon tumor associated-neutrophils (TAN) expressed the cytolytic enzyme granzyme B, which is absent in spleen or blood circulating neutrophils. This TAN population was also found into tumors of patients with colon cancer. Tumor neutrophil infiltration was correlated with an increase of chemokines known to attract neutrophils in both rat models and patients. These cells were involved in a Lipid A analog-mediated colon tumor regression. Mechanistically, treating the rats with the Lipid A analog triggered granzyme B release from neutrophils in tumor cell vicinity, which was correlated to tumor regression. Alteration of granzyme B function in tumor cells decreased the cytotoxic effect of Lipid A in rat and mouse models. Granzyme B expression in neutrophils could be induced by the lipid A analog but also by some of the cytokines that were detected in the tumor microenvironment. These results identify a subpopulation of neutrophils expressing granzyme B that can act as a key player of lipid A-mediated colon cancer regression in rat and mouse models and the molecular mechanisms involved may provide novel approaches for human therapeutic intervention.

TLR4/IFNγ pathways induce tumor regression via NOS II-dependent NO and ROS production in murine breast cancer models.

Toll-like receptor (TLR) 4 agonists have emerged as a new group of molecules used for cancer therapy. They have been exploited to enhance the immunogenicity of current chemotherapeutic regimens. However, their effects on cancer cells remain elusive. Here, we showed that a TLR4 agonist, namely a synthetic lipid A analog (ALA), OM-174, exhibits antitumor effects in several mammary tumor mouse models. We also showed that immune components are involved in such effects, as attested to by the failure of ALA to induce tumor regression or an increase of animal survival in mice knocked-out for interferon γ (IFNγ) or TLR4. TLR4 and IFNγ receptor (INFR2) expressed by cancer cells are involved in the antitumor efficacy of ALA since this last did not inhibit tumor growth in mice bearing a tumor but lacking TLR4 or IFNγ receptor 2 (IFNR2). Mechanistic investigations revealed that nitric oxide (NO), superoxide and peroxynitrite produced by uncoupling of inducible NO synthase (NOS II) in cancer cells are key mediators of ALA and IFNγ-mediated tumor growth inhibition. We present here a comprehensive picture of tumor cell death induction, in vivo and in vitro, by immunotherapy and for the first time the involvement of the TLR4/IFNγ/NOS II pathway in immunotherapy was investigated.

Oct-2 forms a complex with Oct-1 on the iNOS promoter and represses transcription by interfering with recruitment of RNA PolII by Oct-1.

Oct-1 (POU2f1) and Oct-2 (POU2f2) are members of the POU family of transcription factors. They recognize the same DNA sequence but fulfil distinct functions: Oct-1 is ubiquitous and regulates a variety of genes while Oct-2 is restricted to B-cells and neurones. Here we examine the interplay and regulatory mechanisms of these factors to control the inducible nitric oxide synthase (iNOS, NOS2). Using two breast cancer cell lines as a comparative model, we found that MCF-7 express iNOS upon cytokine stimulation while MDA-MB-231 do not. Oct-1 is present in both cell lines but MDA-MB-231 also express high levels of Oct-2. Manipulation of Oct-2 expression in these cell lines demonstrates that it is directly responsible for the repression of iNOS in MDA-MB-231. In MCF-7 cells Oct-1 binds the iNOS promoter, recruits RNA PolII and triggers initiation of transcription. In MDA-MB-231 cells, both Oct-1 and Oct-2 bind the iNOS promoter, forming a higher-order complex which fails to recruit RNA PolII, and as a consequence iNOS transcription does not proceed. Unravelling the mechanisms of transcription factor activity is paramount to the understanding of gene expression patterns that determine cell behaviour.

H89 enhances the sensitivity of cancer cells to glyceryl trinitrate through a purinergic receptor-dependent pathway.

High doses of the organic nitrate glyceryl trinitrate (GTN), a nitric oxide (NO) donor, are known to trigger apoptosis in human cancer cells. Here, we show that such a cytotoxic effect can be obtained with subtoxic concentrations of GTN when combined with H89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide.2HCl. This synergistic effect requires the generation of reactive oxygen species (ROS) from H89 and NO from GTN treatment that causes cGMP production and PKG activation. Furthermore, the GTN/H89 synergy was attenuated by inhibition of P2-purinergic receptors with suramin and competition with ATP/UDP. By down-regulating genes with antisense oligonucleotides, P2-purinergic receptors P2X3, P2Y1, and P2Y6 were found to have a role in creating this cytotoxic effect. Thus, H89 likely acts as an ATP mimetic synergizing with GTN to trigger apoptosis in aggressive cancer cells.

Senescence of tumor cells induced by oxaliplatin increases the efficiency of a lipid A immunotherapy via the recruitment of neutrophils.

Management of advanced colorectal cancer is challenging due to the lack of efficient therapy. The lipid A, OM-174 exhibited antitumor activity in colorectal cancer. We explored the anticancer efficacy of this compound in rats bearing large colorectal tumors in combination with the platinum derivative drugs oxaliplatin and cisplatin. While each drug used alone exhibited partial antitumor activity, sequential treatment with oxaliplatin or cisplatin for one week followed by lipid A injections induced a great regression of colorectal tumors, with more than 95% of rats cured from their tumors. This potent antitumor efficacy of the combined treatments was correlated to the sequential induction of cellular senescence by oxaliplatin, and of apoptosis, mainly triggered by the lipid A. Moreover, a recruitment of tumor-associated neutrophils with N1 phenotype as attested by the expression of inducible nitric oxide synthase was observed with combination of oxaliplatin and lipid A. Neutrophil recruitment within tumor microenvironment was due to oxaliplatin and lipid A-dependent release of neutrophil specific chemoattractants such as cxcl1 and 2. However the N1 phenotype is only dependent of lipid A treatment. These results suggest that the combination of chemotherapy with an immunotherapy is a promising approach to treat patients with advanced colorectal cancer.

Phase I study of OM-174, a lipid A analogue, with assessment of immunological response, in patients with refractory solid tumors.

BACKGROUND: Lipids A, the lipophilic partial structure of lipopolysaccharides, induce regression of several tumor types in animal models. Rather than exerting direct cytotoxic effect, these compounds trigger the immune system which in turn stimulates secretion of cytokines, and activates the inducible nitric oxide synthase, as well as immune cell infiltration of tumors. OM-174 is an analogue of lipid A with dual action on Toll-like receptors 2 and 4. In an experimental model of peritoneal carcinomatosis induced in BDIX rats by intraperitoneal injection of syngeneic PROb colon cancer cells, it induced a complete regression of tumors. The present phase I trial was conducted to determine the maximum tolerated dose, the recommended phase II dose and biological response associated with OM-174 administered as intravenous infusion. METHODS: Patients received OM-174 twice weekly for a total of 5, 10 or 15 injections of either 600, 800 or 1000 µg/m(2). Blood samples for pharmacokinetic analysis and cytokine dosages were collected. NK cells activity and Toll-like receptors 4 polymorphism analysis were also performed. RESULTS: Seventeen patients were included. The highest dose administered was 1000 µg/m(2) repeated in 15 injections. The most common toxicities were a chills, fever, nausea/vomiting, diarrhea, fatigue and headache. No patient experienced haematological side effects. As no dose limiting toxicity was observed, despite a grade 3 respiratory complication, the maximal tolerated dose and recommended dose were not established. Three patients exhibited disease stabilization with a mean duration of 4 months. Pharmacokinetic profile of OM-174 was characterized by a low distribution volume and clearance. Analysis of TLR 4 polymorphysm showed that most (16/17) patients carried the wild type alleles. A progressive increase in NK cell number and activity was observed only in patients receiving 1000 µg/m(2) of OM-174. A peak of IL-8 and IL-10 concentrations were observed after each OM-174 injection. Peaks of TNF-alpha and IL-6 concentrations were detected after the first infusion and decreased progressively suggesting tolerance. CONCLUSION: OM-174 therapy was well tolerated at biologically active concentrations. Whereas the recommended dose was not determined, further studies are planned in combination with chemotherapy as animal models suggest a strong synergistic antitumor effect. TRIAL REGISTRATION: NCT01800812 (ClinicalTrials.gov Identifier).

Oct-1 cooperates with the TATA binding initiation complex to control rapid transcription of human iNOS.

Expression of the human inducible nitric oxide synthase (hiNOS) is generally undetectable in resting cells, but stimulation by a variety of signals including cytokines induces transcription in most cell types. The tight transcriptional regulation of the enzyme is a complex mechanism many aspects of which remain unknown. Here, we describe an octamer (Oct) element in hiNOS proximal promoter, located close to the TATA box. This site constitutively binds Oct-1 and its deletion abrogates cytokine-induced transcription, showing that it is indispensable though not sufficient for transcription. Increasing the distance between Oct and the TATA box by inserting inert DNA sequence inhibits transcription, and footprinting of this region shows no other protein binding in resting cells, suggesting an interaction between the two complexes. Chromatin immunoprecipitation assays detect the presence of Oct-1, RNA polymerase II and trimethyl K4 histone H3 on the proximal promoter in resting cells, confirming that the gene is primed for transcription before stimulation. RT-PCR of various fragments along the hiNOS gene shows that transcription is initiated in resting cells and this is inhibited by interference with Oct-1 binding to the proximal site of the promoter. We propose that, through interaction with the initiation complex, Oct-1 regulates hiNOS transcription by priming the gene for the rapid response required in an immune response.

S-nitrosylation of the death receptor fas promotes fas ligand-mediated apoptosis in cancer cells.

BACKGROUND & AIMS: Fas belongs to the family of tumor necrosis factor receptors which induce apoptosis. Many cancer cells express Fas but do not undergo Fas-mediated apoptosis. Nitric oxide reverses this resistance by increasing levels of Fas at the plasma membrane. We studied the mechanisms by which NO affects Fas function. METHODS: Colon and mammary cancer cell lines were incubated with the NO donor glyceryl trinitrate or lipid A; S-nitrosylation of Fas was monitored using the biotin switch assay. Fas constructs that contained mutations at cysteine residues that prevent S-nitrosylation were used to investigate the involvement of S-nitrosylation in Fas-mediated cell death. Apoptosis was monitored according to morphologic criteria. RESULTS: NO induced S-nitrosylation of cysteine residues 199 and 304 in the cytoplasmic part of Fas. In cancer cells that overexpressed wild-type Fas, S-nitrosylation induced Fas recruitment to lipid rafts and sensitized the cells to Fas ligand. In cells that expressed a mutant form of Fas in which cysteine 304 was replaced by valine residue, NO-mediated translocation of Fas to lipid rafts was affected and the death-inducing signal complex and synergistic effect of glyceryl trinitrate-Fas ligand were inhibited significantly. These effects were not observed in cells that expressed Fas with a mutation at cysteine 199. CONCLUSIONS: We identified post-translational modifications (S-nitrosylation of cysteine residues 199 and 304) in the cytoplasmic domain of Fas. S-nitrosylation at cysteine 304 promotes redistribution of Fas to lipid rafts, formation of the death-inducing signal complex, and induction of cell death.

Lipid A-induced responses in vivo.

The lipid A analogs used in preclinical studies and clinical trials are not naturally-occurring forms of lipid A; they are synthetic molecules produced to be less toxic than lipid A itself and they do not reproduce the effects of natural lipid A molecules especially in vivo. The responses induced by lipid A analogs are summarized in this chapter: their fate in the blood stream and their toxicity as well as the lipid A tolerance and the tumor immune responses they induce. Lipid A is not found in the mammalian organism under normal circumstances so its use in cancer therapy raises important questions as to its different effects in vivo and its toxicity, particularly in cancer patients. Lipid A has to be injected intravenously (i.v.) to study its effects. Injections of chemically synthesized lipid A in humans and in animals produce sepsis symptoms, such as tachycardia, tachypnea, hyper or hypothermia and leukocytosis or leukopenia. Similar manifestations are observed after injection of purified lipopolysaccharide (LPS), which is why lipid A is usually thought of as the active part of LPS. While lipid A injection is therefore expected to induce reactions similar to septic shock, the lipid A molecules used to treat cancer are not natural forms but analogs, produced by chemical synthesis or genetic engineering, specifically selected for their low toxicity. The in vivo effects of such low-toxicity lipid A analogs are summarized in this chapter.

Lipid A in cancer therapies preclinical results.

Studies in animal models showed that the antitumoral effect of LPS and of their biologically active moiety, lipid A, is indirect and relies on the induction of an immune response both innate and specific, leading to cytokine production. They also affect tumor development by inhibiting tumor blood flow and induce necrosis as well as apoptosis of tumor cells. Lipids A have been tested in animals, either alone or as adjuvant in therapeutic vaccines. The efficacy of treatments depends on the type of molecule and on the protocol. In general, increased survival was obtained, accompanied in some cases by tumor regression and cure.

Innate immune response triggered by triacyl lipid A is dependent on phospholipid transfer protein (PLTP) gene expression.

Hexaacyl lipopolysaccharide (LPS) aggregates in aqueous media, but its partially deacylated lipid A moiety forms monomers with weaker toxicity. Because plasma phospholipid transfer protein (PLTP) transfers hexaacyl LPS, its impact on metabolism and biological activity of triacyl lipid A in mice was addressed. Triacyl lipid A bound readily to plasma high-density lipoproteins (HDLs) when active PLTP was expressed [HDL-associated lipid A after 4.5 h: 59.1+/-16.0% of total in wild-type (WT) vs. 32.5+/-10.3% in PLTP-deficient mice, P<0.05]. In the opposite to hexaacyl LPS, plasma residence time of lipid A was extended by PLTP, and proinflammatory cytokines were produced in higher amounts in WT than PLTP(-/-) mice (remaining lipid A after 8 h: 53+/-12 vs. 35+/-7%, and IL6 concentration after 4.5 h: 45.5+/-5.9 vs. 14.6+/-7.8 ng/ml, respectively; P<0.05 in all cases). After 1 wk, onset of B16-induced melanoma was observed in only 30% of lipid A-treated WT mice, whereas >80% of the untreated WT, untreated PLTP-deficient, or lipid A-treated PLTP-deficient animals bore tumors (P<0.05 in all cases). It is concluded that PLTP is essential in mediating the association of triacyl lipid A with lipoproteins, leading to extension of its residence time and to magnification of its proinflammatory and anticancer properties.

Post-translational modifications induced by nitric oxide (NO): implication in cancer cells apoptosis.

Post-translational modifications of proteins can regulate the balance between survival and cell death signals. It is increasingly recognized that nitric oxide (NO) and reactive oxygen species (ROS)-induced post-translational modifications could play a role in cell death. This review provides an introduction of current knowledge of NO proteins modifications promoting or inhibiting cell death with special attention in cancer cells.

Nitric oxide-induced resistance or sensitization to death in tumor cells.

This report summarizes the present state of our knowledge pertaining to the NO-induced resistance or sensitization of tumor cell death. The effects of NO and its synergy with members of the TNF family, with cytotoxic drugs, and with ionizing radiations have been investigated. The dual effect of NO-induced resistance or sensitization and the underlying molecular mechanisms are discussed.

Cancer relapse under chemotherapy: why TLR2/4 receptor agonists can help.

Liver or lung metastases usually relapse under chemotherapy. Such life-threatening condition urgently needs new, systemic anticancer compounds, with original and efficient mechanisms of action. In B16 melanoma mice treated with cyclophosphamide, D'Agostini et al. [D'Agostini, C., Pica, F., Febbraro, G., Grelli, S., Chiavaroli, C., Garaci, E., 2005. Antitumour effect of OM-174 and Cyclophosphamide on murine B16 melanoma in different experimental conditions. Int. Immunopharmacol. 5, 1205-1212.] recently found that OM-174, a chemically defined Toll-like receptor(TLR)2/4 agonist, reduces tumor progression and prolongs survival. Here we review 149 articles concerning molecular mechanisms of TLR2/4 agonists, alone or in combination with chemotherapy. It appears that TLR2/4 agonists induce a well controlled tumor necrosis factor-alpha (TNF-alpha) secretion, at plasma levels known to permeabilize neoangiogenic tumor vessels to the passage of cytotoxic drugs. Moreover, TLR2/4 agonists induce inducible nitric oxide synthase (iNOS) expression, and nitric oxide is able to induce apoptosis of chemotherapy-resistant tumor cell clones. Finally, TLR2/4-stimulation activates dendritic cell traffic and its associated tumor-specific, cytotoxic T-cell responses. Therefore, parenteral TLR2/4 agonists seem promising molecules to prolong survival in cancer patients who relapse under chemotherapy.

Nitric oxide-induced down-regulation of beta-catenin in colon cancer cells by a proteasome-independent specific pathway.

BACKGROUND & AIMS: We have previously reported that nitric oxide could induce the death of colon cancer cells. Because an inappropriate activation of beta-catenin has been associated with intestinal cell malignant transformation, we explored whether nitric oxide could affect beta-catenin expression and function. METHODS: Human colon cancer cell lines were treated with the nitric oxide donor glyceryl trinitrate (GTN) before analyzing beta-catenin expression by immunofluorescence, immunoblotting, and immunoprecipitation methods and its transcriptional activity using a luciferase reporter gene driven by a T-cell factor-responsive promotor. RESULTS: GTN induces beta-catenin degradation and down-regulates its transcriptional activity in colon cancer cells. This effect is preceded by GTN-induced tyrosine nitration of beta-catenin, together with its dephosphorylation on serine 33, 37, and 45 and threonine 41. GTN-induced beta-catenin degradation involves proteases that are sensitive to a broad-spectrum caspase inhibitor, z-VAD-fmk, and to serine protease inhibitors N-tosyl-L-phenylalaline chloromethyl ketone (TPCK) and [4-(2-aminoethyl)-benzenesulfonylfluoride] (AEBSF), whereas the ubiquitin/proteasome pathway is not involved. Interestingly, only TPCK and AEBSF restore beta-catenin transcriptional activity and preserve beta-catenin nuclear localization in GTN-treated colon cancer cells. CONCLUSIONS: Exposure of colon cancer cells to nitric oxide unraveled a so-far-unidentified mechanism of beta-catenin regulation. The protein is nitrated and dephosphorylated, and its transcriptional activity is reduced through degradation by a TPCK and AEBSF-sensitive protease.

Plasticity in cell defence: access to and reactivity of critical protein residues and DNA response elements.

Cellular and whole organ defence against pathogenic or chemical challenge is manifest as an adaptive response. Where appropriate, this may lead to induction of a cellular defence programme, thereby enhancing cell survival. When the challenge is overwhelming, the defence is breached and a switch is made to yield cell death, either by apoptosis or necrosis. Thus, a cell will defend itself where possible, but in extremis, it may recognise the futility of its resistance and allow itself to die. Transcription factor activation and access to the DNA regulatory elements that control a particular pattern of expression of defence genes is a major issue that may ultimately decide the fate of a cell in a changed environment. It is possible to visualise the access to the nucleus and to the genome, of paradigm gene loci or transcription factors, using a number of molecular techniques such as chromatin immunoprecipitation, in vivo footprinting and live/whole cell imaging. These methods are informative as to the array of transcription factors that may regulate a given gene, as well as the transitory nature of the transcriptional activation. The initial triggering of active transcription factor complexes typically occurs within the cytoplasm of the cell. Protein-protein interactions and signal transduction pathways, elucidated using a classical molecular genetics approach, have long been recognised as pivotal to the initial control of the levels and activity of transcription factors. We can now visualise modifications in critical residues of transcription factors and regulators during cellular response to chemical stress. These modifications may yield enhanced or repressed activity of transcription factors, they may be non-covalent or covalent, and they may occur in response to a variety of classes of chemicals. Such promiscuous signalling can provide plasticity in the cellular response to a wide array of chemical agents.

Evaluation of the interaction between TGF beta and nitric oxide in the mechanisms of progression of colon carcinoma.

It is recognised that stromal cells determine cancer progression. We have previously shown that active TGFbeta produced by rat colon carcinoma cells modulated NO production in rat endothelial cells. To elucidate the role of TGFbeta and NO in the mechanisms of interaction of colon carcinoma cells with stromal cells and in cancer progression, we transfected REGb cells, a regressive colon carcinoma clone secreting latent TGFbeta, with a cDNA encoding for a constitutively-secreted active TGFbeta. Out of 20 injected rats only one tumour progressed, which was resected and sub-cultured (ReBeta cells). ReBeta cells secreted high levels of active TGFbeta. The adhesive properties of REGb and Rebeta cells to endothelial cells were similar, showing that the secretion of active TGFbeta is not involved in tumour cell adhesion to endothelial cells. ReBeta, but not REGb, cell culture supernatants inhibited cytokine-dependent NO secretion by endothelial cells, but inhibition of NO production was similar in co-cultures of REGb or ReBeta cells with endothelial cells. Therefore, secretion of active TGFbeta regulated endothelial NO synthase activity when tumour cells were distant from, but not in direct contact with, endothelial cells. However, only ReBeta cells inhibited cytokine-dependent secretion of NO in coculture with macrophages, indicating that the active-TGFbeta-NO axis confers an advantage for tumour cells in their interaction with macrophages rather than endothelial cells in cancer progression.

Progesterone enhances cytokine-stimulated nitric oxide synthase II expression and cell death in human breast cancer cells.

The presence of hormone receptors is related to survival outcome in breast cancer. Previous results from our laboratory established a correlation between the presence of nitric oxide synthase II (NOSII) and nitric oxide (NO) production with progesterone receptors in a series of human breast tumours. Furthermore, this was directly related to a lower tumour grade and a lower proliferation rate of the tumour cells. To examine these results in further detail, the effect of progesterone (Pg) and 17beta-oestradiol (E2) on NOSII expression was analysed in the human breast cancer cell line MCF-7. By Northern blot and promoter activity, we show that a cytokine mix (TNF-alpha, IL-beta, and IFN-gamma) induces NOSII transcription after 6 h stimulation. In the absence of cytokines, neither hormone affects NOSII expression. However, Pg but not E2, enhances cytokine-induced NOSII transcription as well as NO synthesis, mainly by cooperation with gamma-interferon. The increase in NO accumulation in the media induced by addition of Pg to the cytokine treatment significantly increases cell death, mainly accounted for by apoptosis, as compared to the effect of cytokines alone. Our findings help clarify the role of steroid hormones in NOSII expression as well as the effect on cell viability and may suggest novel approaches towards hormonotherapy and the treatment of cancer.