Prostaglandin E(2) (PGE(2)) and epinephrine act directly on nociceptors to produce mechanical hyperalgesia through protein kinase A (PKA) alone or through a combination of PKA, protein kinase C epsilon (PKCepsilon), and extracellular signal-regulated kinase (ERK), respectively. Disruptors of the cytoskeleton (microfilaments, microtubules, and intermediate filaments) markedly attenuated the hyperalgesia in rat paws caused by injection of epinephrine or its downstream mediators. In contrast, the hyperalgesia induced by PGE(2) or its mediators was not affected by any of the cytoskeletal disruptors. These effects were mimicked in vitro, as measured by enhancement of the tetrodotoxin-resistant sodium current. When PGE(2) hyperalgesia was shifted to dependence on PKCepsilon and ERK as well as PKA, as when the tissue is "primed" by prior treatment with carrageenan, it too became dependent on an intact cytoskeleton. Thus, inflammatory mediator-induced mechanical hyperalgesia was differentially dependent on the cytoskeleton such that cytoskeletal dependence correlated with mediation by PKCepsilon and ERK.

1 Transfection of the pre-monomyelocytic U937 cell line with a plasmid coding for full-length annexin 1 (ANX1, 347 amino acid) leads to cell death by promoting apoptosis. In addition, overexpression of the N-terminal and the ®rst domain of the protein (144 amino acids, clone ANX1-S), which does not contain the Ca 2+ binding sites, gives susceptibility to cell apoptosis following activation by either 5 ng ml 71 tumour necrosis factor (TNF)-a or 1 ± 40 mg ml 71 etoposide. This was demonstrated by using the¯uorescent labelled annexin V, cell cycle and nuclear staining analyses. 2 Transfection with an empty plasmid (clone CMV) or with a plasmid carrying the cDNA antisense for ANX1 (clone ANX1-AS) did not alter U937 cells to the degree of apoptosis promoted by either stimulant. 3 Treatment of CMV U937 cells with TNF-a increased ANX1 mRNA and protein expression in a time-dependent manner, with maximal increases at 3 and 6 h, respectively. 4 Clone ANX1-S showed higher constitutive (more than 2 fold) and activated caspase-3 activity, associated with higher phospholipase A 2 (PLA 2 ) activity (in the region of +50 ± 100%), whereas expression of cytosolic PLA 2 Bax and Bcl-2 were similar in all cell clones, as determined by Western blotting. 5 In conclusion, this study demonstrates a complex regulatory role of cell apoptosis for ANX1, at least with regards to cells of the myelo-monocytic lineage.

Short peptide sequences that are able to transport molecules across the cell membrane have been developed as tools for intracellular delivery of therapeutic molecules. This work describes a novel family of cell-penetrating peptides named Vectocell peptides [also termed DPVs (Diatos peptide vectors)]. These peptides, originating from human heparin binding proteins and/or anti-DNA antibodies, once conjugated to a therapeutic molecule, can deliver the molecule to either the cytoplasm or the nucleus of mammalian cells. Vectocell peptides can drive intracellular delivery of molecules of varying molecular mass, including full-length active immunoglobulins, with efficiency often greater than that of the well-characterized cell-penetrating peptide Tat. The internalization of Vectocell peptides has been demonstrated to occur in both adherent and suspension cell lines as well as in primary cells through an energy-dependent endocytosis process, involving cell-membrane lipid rafts. This endocytosis occurs after binding of the cell-penetrating peptides to extracellular heparan sulphate proteoglycans, except for one particular peptide (DPV1047) that partially originates from an anti-DNA antibody and is internalized in a caveolar independent manner. These new therapeutic tools are currently being developed for intracellular delivery of a number of active molecules and their potentiality for in vivo transduction investigated.

We have used a transgenic animal model, which constitutively develops hepatocarcinoma (Antithrombin III SV40 T large Antigen: ASV), to study the involvement of Annexin 1 (ANX1) in liver regeneration and malignant transformation. Primary hepatocytes isolated from normal mice did not express ANX1. In contrast, ANX1 was strongly expressed in hepatocytes of transgenic mice during constitutive development of hepatocarcinoma. In ASV transgenic mice, an elevated ANX1 level preceded the appearance of the tumor, indicating that it could be a good marker in the diagnosis of cancer. One-third hepatectomy in normal mice resulted in stimulation of ANX1 synthesis and phosphorylation. This upregulation correlated with increased synthesis of EGF and consequently with increased phosphorylation of the EGF receptor (EGF-R). Stable transfection of a hepatocyte cell line derived from ASV transgenic mice (mhAT2) with antisense complementary DNA for ANX1 reduced the proliferation rate as well as cytosolic phospholipase A 2 (cPLA 2 ) activity. Thus, ANX1 expression and phosphorylation could be a factor implicated in liver regeneration and tumorigenesis, either through modulation of cPLA 2 activity or EGF-R function. (HEPATOLOGY 2000;31:371-380.)Annexin 1 (ANX1) is a member of a multigene family of Ca 2ϩ /phospholipid binding proteins and a major substrate for the epidermal growth factor (EGF) receptor kinase. 1 We have shown that ANX1, is transcriptionally regulated by interleukin-6 (IL-6) and phorbol myristate acetate (PMA) in human lung adenocarcinoma cell (A 549) through the induction of C/EBP␤ transcription factor. Based on these data we proposed a dual role for ANX1 in cellular differentiation 2 and as a new acute-phase protein. 3 These results prompted us to investigate the role of ANX1 in the liver. While we were carrying these experiments, ANX1 was reported to be strongly expressed in human hepatocellular (hcc) carcinoma cells, suggesting that its induction in the liver could be associated with malignant transformation. 4,5 We therefore addressed the question of the role of ANX1 in liver transformation.Recently, transgenic mice (ASV) expressing the SV40 early region coding for large T antigen under the control of the liver-specific human antithrombin III gene promoter have been created in our laboratory to study oncogenic transformation on hepatocyte function. 6 Phenotypically, the animals present a hepatocellular carcinoma, with a neoplasia at 3 to 9 weeks and later (after tumor development) a severe dysplasia, an augmentation of hepatocytes in S phases, an important cytolysis (necrosis), and an early apoptosis (2.5%-5%). Up to 25 weeks no major difference was obvious between the liver weight of transgenic (ASV) and control mice. After that, liver weight progressively increased reaching sevenfold the normal liver weight at the terminal stage of hcc. Eight old mice die for a general disorder and an important cachexia. 6 Using this model as paradigm for our study of the role of ANX1 in liver, we now report that ANX1 is strongly ...

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Annexin 1 (An 1), a phospholipid and calcium binding protein, is strongly expressed in differentiated U 937 cells. In attempting to correlate the expression of An 1 with phospholipase A2 (PLA2) activity, U 937 cells were stably transfected both with a Sense and Antisense cDNA for An 1. PLA2 activity was measured by Flow cytometry analysis utilizing the bis‐Bodipy‐C11‐PC fluorescent probe. U 937 cells stably transfected with the sense or antisense vectors were differentiated for 24 h with phorbol 12‐myristate 13‐acetate (PMA, 6 ng ml−1). Both in undifferentiated and differentiated cells, the Antisense clone (36.4 AS) showed consistently higher PLA2 activity than the control Sense clone (15 S). Since the fluorescent probe measures the total PLA2 activity, we used two different stimuli, PMA: (100 ng ml−1) or lipopolysaccharide (LPS, 10 ng ml−1), and two different inhibitors, to discriminate the PLA2 involved (namely arachidonyl trifluoromethyl ketone or AACOCF3, which is specific for the cytosolic PLA2, and SB 203347 specific for the secretory PLA2). In the Antisense clone the inhibitory effect of AACOCF was stronger [68%, P<0.025] than in the Sense, which may reflect the lower endogenous level of An 1 present in the cells. On the contrary, the inhibitory effect of SB 203347 [60% of inhibition] was identical in both clones. Since cPLA2 activity is correlated with its phosphorylation, Western and shift blot analysis were performed. They did not show any significative difference between the phosphorylated and non phosphorylated form of the enzyme in both the differentiated or not, Sense and Antisense clones. Furthermore the tyrosine phosphorylation analysis of An 1 showed that less than 10% of An 1 was phosphorylated irrespective of PMA presence or absence. From the pattern of inhibition observed, we propose that the endogenous unphosphorylated form of An 1 may act intracellularly to block the activity of a cytosolic PLA2. British Journal of Pharmacology (1998) 124, 1675–1683; doi:

Annexin 1 (ANXA1) is a calcium-binding protein endowed with anti-inflammatory properties. Using an extra-hepatic system, we showed that interleukin (IL)-6 regulates ANXA1 expression at the transcriptional level. The purpose of this study was to determine whether ANXA1 synthesis was modulated by IL-6 during experimental inflammation. We have compared liver ANXA1 expression during systemic and localized inflammatory reaction, using lipopolysaccharide (LPS) and turpentine. LPS treatment strongly induced ANXA1 expression in the liver of wild-type (WT) animals (+600%) whereas a modest increase (+60%) was measured in IL-6 knockout (KO) animals. Turpentine treatment did not affect the expression of ANXA1 in either animal type. LPS enhanced serum corticosteroid levels equally in WT and IL-6 KO mice, whereas higher tumor necrosis factor (TNF)-alpha and IL-1beta levels were released in IL-6 KO animals. Injection of mouse recombinant IL-6 to IL-6 KO animals before LPS or TNF-alpha challenge, replenished ANXA1 liver synthesis to that of WT animals. Exogenous ANXA1 but not ANXA5, administered to IL-6 KO mice before LPS challenge inhibited TNF-alpha release. We propose that ANXA1 acts as a novel acute phase protein, which is controlled in the liver by TNF-alpha and IL-6, and which may contribute to the resolution of systemic endotoxemia through a negative feedback on TNF-alpha release.

Purpose: Irinotecan is a prodrug converted to the active cytotoxic molecule SN38 predominantly by the action of liver carboxylesterases. The efficacy of irinotecan is limited by this hepatic activation that results in a low conversion rate, high interpatient variability, and dose-limiting gastrointestinal toxicity. The purpose of this study was to evaluate a novel peptidic prodrug of SN38 (DTS-108) developed to bypass this hepatic activation and thus reduce the gastrointestinal toxicity and interpatient variability compared with irinotecan. Experimental Design: SN38 was conjugated to a cationic peptide (Vectocell) via an esterase cleavable linker.The preclinical development plan consisted of toxicity and efficacy evaluation in a number of different models and species. Results: The conjugate (DTS-108) is highly soluble, with a human plasma half-life of 400 minutes in vitro. Studies in the dog showed that DTS-108 liberates significantly higher levels of free SN38 than irinotecan without causing gastrointestinal toxicity. In addition, the ratio of the inactive SN38-glucuronide metabolite compared with the active SN38 metabolite is significantly lower following DTS-108 administration, compared with irinotecan, which is consistent with reduced hepatic metabolism. In vivo efficacy studies showed that DTS-108 has improved activity compared with irinotecan. A significant dose-dependent antitumoral efficacy was observed in all models tested and DTS-108 showed synergistic effects in combination with other clinically relevant therapeutic agents. Conclusions: DTS-108 is able to deliver significantly higher levels of SN38 than irinotecan, without the associated toxicity of irinotecan, resulting in an increased therapeutic window for DTS-108 in preclinical models. These encouraging data merit further preclinical and clinical investigation.Irinotecan is an effective chemotherapeutic agent that is widely prescribed for advanced colorectal cancer as a first-or secondline treatment. Irinotecan has also been shown to be active in gastric cancer, non -small cell lung cancer, and small-cell lung cancer, alone or in combination with other cytotoxic agents (1). Currently, irinotecan is used in combination with 5-fluorouracil (5-FU) in first-line treatment for metastatic colorectal cancer (2). Irinotecan is also used with other agents, including the anti -vascular endothelial growth factor antibody bevacizumab (Avastin; refs. 3,4). Following administration of irinotecan, the active metabolite SN38 (7-ethyl-10-hydroxycamptothecin) is formed by the action of carboxylesterases that are predominantly present in the liver (5, 6). SN38 is a topoisomerase I inhibitor with an activity a thousand times greater than irinotecan, but that cannot be administered directly as it is highly insoluble (1).Several limitations to the clinical use of irinotecan arise due to its mechanism of activation, metabolism, and elimination. The first limitation is caused by the complexity of irinotecan metabolism, which results in high interpatient variabil...