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PDF file - 635K, Supplementary Figure S1. Genotypes included in the combinatorial genetic matrix. PTEN or RB1 were silenced by shRNA in isogenic KI cells.

XLSX file - 144K, Supplementary table S1. List of compounds employed in the present work. Supplementary Table S2. List of t-tests p values comparing the responses of the different mutant cell lines to a given drug concentration with the response of the WT Scramble control cells. Supplementary Table S3. List of the drugs included in the library and tested on HME-1 cell lines.

A critical step toward defining tailored therapy in patients with cancer is the identification of genetic interactions that may impair—or boost—the efficacy of selected therapeutic approaches. Cell models able to recapitulate combinations of genetic aberrations are important to find drug–genotype interactions poorly affected by the heterogeneous genetics of human tumors. In order to identify novel pharmacogenomic relationships, we employed an isogenic cell panel that reconstructs cancer genetic scenarios. We screened a library of 43 compounds in human hTERT-HME1 epithelial cells in which PTEN or RB1 were silenced in combination with the targeted knockin of cancer-associated mutations in EGFR, KRAS, BRAF, or PIK3CA oncogenes. Statistical analysis and clustering algorithms were applied to display similar drug response profiles and mutation-specific patterns of activity. From the screen, we discovered that proteasome inhibitors show selectivity toward BRAF V600E–mutant cells, irrespective of PTEN or RB1 expression. Preferential targeting of BRAF-mutant cells by proteasome inhibitors was corroborated in a second BRAF V600E isogenic model, as well as in a panel of colorectal cancer cell lines by the use of the proteasome inhibitor carfilzomib. Notably, carfilzomib also showed striking in vivo activity in a BRAF-mutant human colorectal cancer xenograft model. Vulnerability to proteasome inhibitors is dependent on persistent BRAF signaling, because BRAF V600E blockade by PLX4720 reversed sensitivity to carfilzomib in BRAF-mutant colorectal cancer cells. Our findings indicated that proteasome inhibition might represent a valuable targeting strategy in BRAF V600E–mutant colorectal tumors. Mol Cancer Ther; 12(12); 2950–61. ©2013 AACR.

The file lists Supplementary Materials and Methods and relative References.

Although recent clinical trials of BRAF inhibitor combinations have demonstrated improved efficacy in BRAF-mutant colorectal cancer, emergence of acquired resistance limits clinical benefit. Here, we undertook a comprehensive effort to define mechanisms underlying drug resistance with the goal of guiding development of therapeutic strategies to overcome this limitation. We generated a broad panel of BRAF-mutant resistant cell line models across seven different clinically relevant drug combinations. Combinatorial drug treatments were able to abrogate ERK1/2 phosphorylation in parental-sensitive cells, but not in their resistant counterparts, indicating that resistant cells escaped drug treatments through one or more mechanisms leading to biochemical reactivation of the MAPK signaling pathway. Genotyping of resistant cells identified gene amplification of EGFR, KRAS, and mutant BRAF, as well as acquired mutations in KRAS, EGFR, and MAP2K1. These mechanisms were clinically relevant, as we identified emergence of a KRAS G12C mutation and increase of mutant BRAF V600E allele frequency in the circulating tumor DNA of a patient at relapse from combined treatment with BRAF and MEK inhibitors. To identify therapeutic combinations capable of overcoming drug resistance, we performed a systematic assessment of candidate therapies across the panel of resistant cell lines. Independent of the molecular alteration acquired upon drug pressure, most resistant cells retained sensitivity to vertical MAPK pathway suppression when combinations of ERK, BRAF, and EGFR inhibitors were applied. These therapeutic combinations represent promising strategies for future clinical trials in BRAF-mutant colorectal cancer. Cancer Res; 76(15); 4504–15. ©2016 AACR.

Supplementary Tables S1-S3 - List of drug concentrations to generate resistant cell lines (S1); Drug concentrations applied in the screening depicted in Figure 6 (S2); Primers for gene amplification and sequencing (S3). Supplementary Figure S1. Synergistic activity of targeted therapy combinations in BRAF mutant colorectal cancer cells. Supplementary Figure S2. Amplification of mutant BRAF V600E confers resistance to combined EGFR and MEK targeting in colorectal cancer cells. Supplemental Figure S3. Cytotoxicity induced by ERK inhibition in VACO432 resistant to BRAF combination therapies.

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In the search for new chemical scaffolds able to afford NLRP3 inflammasome inhibitors, we used a pharmacophore-hybridization strategy by combining the structure of the acrylic acid derivative INF39 with the 1-(piperidin-4-yl)1,3-dihydro-2H-benzo[d]imidazole-2-one substructure present in HS203873, a recently identified NLRP3 binder. A series of differently modulated benzo[d]imidazole-2-one derivatives were designed and synthesised. The obtained compounds were screened in vitro to test their ability to inhibit NLRP3-dependent pyroptosis and IL-1β release in PMA-differentiated THP-1 cells stimulated with LPS/ATP. The selected compounds were evaluated for their ability to reduce the ATPase activity of human recombinant NLRP3 using a newly developed assay. From this screening, compounds 9, 13 and 18, able to concentration-dependently inhibit IL-1β release in LPS/ATP-stimulated human macrophages, emerged as the most promising NLRP3 inhibitors of the series. Computational simulations were applied for building the first complete model of the NLRP3 inactive state and for identifying possible binding sites available to the tested compounds. The analyses led us to suggest a mechanism of protein–ligand binding that might explain the activity of the compounds.

[Image: see text] Aldo-keto reductase 1C3 (AKR1C3) is an attractive target in drug design for its role in resistance to anticancer therapy. Several nonsteroidal anti-inflammatory drugs such as indomethacin are known to inhibit AKR1C3 in a nonselective manner because of COX-off target effects. Here we designed two indomethacin analogues by proposing a bioisosteric connection between the indomethacin carboxylic acid function and either hydroxyfurazan or hydroxy triazole rings. Both compounds were found to target AKR1C3 in a selective manner. In particular, hydroxyfurazan derivative is highly selective for AKR1C3 over the 1C2 isoform (up to 90-times more) and inactive on COX enzymes. High-resolution crystal structure of its complex with AKR1C3 shed light onto the binding mode of the new inhibitors. In cell-based assays (on colorectal and prostate cancer cells), the two indomethacin analogues showed higher potency than indomethacin. Therefore, these two AKR1C3 inhibitors can be used to provide further insight into the role of AKR1C3 in cancer.

A number of oxidovanadium(IV) complexes have been reported to display anticancer activity. A theranostic approach, based on the simultaneous observation of both the effect of oxidovanadium(IV) complexes on cell viability and the disclosure of their intracellular fate, is possible by using oxidovanadium(IV) complexes functionalized with fluorescent ligands. In the present study we accomplished the characterization of six oxidovanadium(IV) complexes in conditions close to those employed for in vitro administration. In particular, we investigated the light harvesting properties of such complexes in the presence of a dimethylsulphoxide/aqueous buffer mixture, and we found that one complex exhibits a quantum yield suitable for confocal microscopy investigations. EPR investigations in the same conditions provide information about the presence of ligands' substitution processes. Finally, the electrochemical properties of all complexes were determined by cyclic voltammetry. The overall results show that these complexes exhibit an average stability in solution; EPR data confirm that DMSO enter the first coordination sphere of oxidovanadium(IV) and suggest the occurrence of partial ligand substitution in the dimethylsulphoxide/aqueous buffer mixture.

Although recent clinical trials of BRAF inhibitor combinations have demonstrated improved efficacy in BRAF-mutant colorectal cancer, emergence of acquired resistance limits clinical benefit. Here, we undertook a comprehensive effort to define mechanisms underlying drug resistance with the goal of guiding development of therapeutic strategies to overcome this limitation. We generated a broad panel of BRAF-mutant resistant cell line models across seven different clinically relevant drug combinations. Combinatorial drug treatments were able to abrogate ERK1/2 phosphorylation in parental-sensitive cells, but not in their resistant counterparts, indicating that resistant cells escaped drug treatments through one or more mechanisms leading to biochemical reactivation of the MAPK signaling pathway. Genotyping of resistant cells identified gene amplification of EGFR, KRAS, and mutant BRAF, as well as acquired mutations in KRAS, EGFR, and MAP2K1. These mechanisms were clinically relevant, as we identified emergence of a KRAS G12C mutation and increase of mutant BRAF V600E allele frequency in the circulating tumor DNA of a patient at relapse from combined treatment with BRAF and MEK inhibitors. To identify therapeutic combinations capable of overcoming drug resistance, we performed a systematic assessment of candidate therapies across the panel of resistant cell lines. Independent of the molecular alteration acquired upon drug pressure, most resistant cells retained sensitivity to vertical MAPK pathway suppression when combinations of ERK, BRAF, and EGFR inhibitors were applied. These therapeutic combinations represent promising strategies for future clinical trials in BRAF-mutant colorectal cancer. Cancer Res; 76(15); 4504–15. ©2016 AACR.

High Brassicaceae consumption reduces the risk of developing several cancer types, probably due to high levels of glucosinolates. Extracts from Sinapis nigra L. (S. nigra) and Sinapis alba L. (S. alba) have been obtained from leaves and seeds under different conditions using ethanol/water mixtures because their glucosinolates are well accepted by the food industry. The EtOH/H2O 8:2 mixture gives better yields in glucosinolate amounts from ground seeds, mainly, sinalbin in S. alba and sinigrin in S. nigra. The highest antiproliferative activity in both non-tumor and tumor cell lines was induced by S. alba seeds extract. To evaluate whether the effect of Sinapis species (spp) was only due to glucosinolate content or whether it was influenced by the extracts’ complexity, cells were treated with extracts or glucosinolates, in the presence of myrosinase. Pure sinigrin did not modify cell proliferation, while pure sinalbin was less effective than the extract. The addition of myrosinase increased the antiproliferative effects of the S. nigra extract and sinigrin. Antiproliferative activity was correlated to Mitogen-Activated Protein Kinases modulation, which was cell and extract-dependent. Cell-cycle analysis evidenced a proapoptotic effect of S. alba on both tumor cell lines and of S. nigra only on HCT 116. Both extracts showed good antimicrobial activity in disc diffusion tests and on ready-to-eat fresh salad. These results underline the potential effects of Sinapis spp in chemoprevention and food preservation.

This work proposes a novel approach by which to consistently classify cysteine sites in proteins in terms of their reactivity toward dimethyl fumarate (DMF) and fumarate. Dimethyl fumarate-based drug products have been approved for use as oral treatments for psoriasis and relapsing-remitting multiple sclerosis. The adduction of DMF and its (re)active metabolites to certain cysteine residues in proteins is thought to underlie their effects. However, only a few receptors for these compounds have been discovered to date. Our approach takes advantage of the growing number of known DMF- and fumarate-sensitive proteins and sites to perform analyses by combining the concepts of network theory, for protein structure analyses, and machine-learning procedures. Wide-ranging and previously unforeseen variety is found in the analysis of the neighborhood composition (the first neighbors) of cysteine sites found in DMF- and fumarate-sensitive proteins. Furthermore, neighborhood composition has shown itself to be a network-type attribute that is endowed with remarkable predictive power when distinct classification algorithms are employed. In conclusion, when adopted in combination with other target identification/validation approaches, methods that are based on the analysis of cysteine site neighbors in proteins should provide useful information by which to decipher the mode of action of DMF-based drugs.

A critical step toward defining tailored therapy in patients with cancer is the identification of genetic interactions that may impair—or boost—the efficacy of selected therapeutic approaches. Cell models able to recapitulate combinations of genetic aberrations are important to find drug–genotype interactions poorly affected by the heterogeneous genetics of human tumors. In order to identify novel pharmacogenomic relationships, we employed an isogenic cell panel that reconstructs cancer genetic scenarios. We screened a library of 43 compounds in human hTERT-HME1 epithelial cells in which PTEN or RB1 were silenced in combination with the targeted knockin of cancer-associated mutations in EGFR, KRAS, BRAF, or PIK3CA oncogenes. Statistical analysis and clustering algorithms were applied to display similar drug response profiles and mutation-specific patterns of activity. From the screen, we discovered that proteasome inhibitors show selectivity toward BRAF V600E–mutant cells, irrespective of PTEN or RB1 expression. Preferential targeting of BRAF-mutant cells by proteasome inhibitors was corroborated in a second BRAF V600E isogenic model, as well as in a panel of colorectal cancer cell lines by the use of the proteasome inhibitor carfilzomib. Notably, carfilzomib also showed striking in vivo activity in a BRAF-mutant human colorectal cancer xenograft model. Vulnerability to proteasome inhibitors is dependent on persistent BRAF signaling, because BRAF V600E blockade by PLX4720 reversed sensitivity to carfilzomib in BRAF-mutant colorectal cancer cells. Our findings indicated that proteasome inhibition might represent a valuable targeting strategy in BRAF V600E–mutant colorectal tumors. Mol Cancer Ther; 12(12); 2950–61. ©2013 AACR.

Vanadium compounds are known to display a number of therapeutic effects, namely insulin-mimetic and cardiovascular effects. Evidence of the antiproliferative and proapoptotic activity of a number of vanadyl complexes, together with their low toxicity, establishes these metal compounds as promising antitumoral therapeutic agents. In the present work, we describe the synthesis and full characterization of six new vanadyl complexes with acetylacetonate derivatives bearing asymmetric substitutions on the β-dicarbonyl moiety: the complexes were characterized in the solid state as well as in solution. Our results show that all complexes are in square pyramidal geometry; cis isomers in the equatorial plane are favored in the presence of strongly coordinating solvents. EPR evidence suggests that all complexes are in the bis-chelate form, although in two cases the mono-chelated complex seems to be present as well. Preliminary tests carried out on non-tumor and tumor cell lines show that these complexes are effective in suppressing cell viability and elicit a distinct response of tumor and non-tumor cells.

Summary With the aim to develop natural preservatives displaying also chemopreventive activity, different Commiphora myrrha (Nees) Engl. extracts were studied. Myrrh essential oils, obtained by steam distillation and microwave-assisted hydrodistillation, and several other extracts, obtained by sequential procedures with petroleum ether (PE), ethanol, ethyl acetate and butanol, have been screened for their antioxidant (DPPH· scavenging assay) and antiproliferative activity (on both nontumour and colon cancer cell lines) without previous purification. Considering that the colon cancer cell lines were more sensitive to PE and ethanol extracts, the latter of which showed the highest antioxidant activity (EC50 = 0.160 ± 0.008 mg mL−1), both have been selected for further antibacterial/antifungal activity tests using an antimicrobial diffusion test and a growth inhibition test on salads. Results showed that the ethanol extract possessed the higher antibacterial and antifungal activity. Compared to untreated product, fresh-cut salads treated with these two myrrh extracts displayed a significant lower bacterial growth. Although further investigation is required, these promising results offer hints as how to improve the shelf life of fresh-cut salad.

A main limitation of therapies that selectively target kinase signalling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor (EGFR), is effective in a subset of KRAS wild-type metastatic colorectal cancers. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood. Here we show that molecular alterations (in most instances point mutations) of KRAS are causally associated with the onset of acquired resistance to anti-EGFR treatment in colorectal cancers. Expression of mutant KRAS under the control of its endogenous gene promoter was sufficient to confer cetuximab resistance, but resistant cells remained sensitive to combinatorial inhibition of EGFR and mitogen-activated protein-kinase kinase (MEK). Analysis of metastases from patients who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60% (6 out of 10) of the cases. KRAS mutant alleles were detectable in the blood of cetuximab-treated patients as early as 10 months before radiographic documentation of disease progression. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancers, indicate that the emergence of KRAS mutant clones can be detected non-invasively months before radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance.

Background and purpose: We investigated the ability of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, to modulate expression of ICAM-1 and VCAM-1 in the colon cancer cell line HT29. Experimental approach: We analysed the effect of celecoxib on ICAM-1 and VCAM-1 protein and mRNA expression in HT29 cells. Experiments were performed in the presence of mitogen-activated protein kinases (MAPK) inhibitors to evaluate the involvement of these kinases in this phenomenon. We evaluated adhesion of HT29 cells to FCS-coated plastic wells in the presence of celecoxib or MAPK inhibitors. Furthermore, we studied the effect of celecoxib on apoptosis. Key results: Celecoxib down-regulated ICAM-1 and VCAM-1 expression in HT29 cells in a time- and dose-dependent way. Celecoxib reduced activation of p38 and p55 c-Jun terminal NH2 kinase (JNK) MAPKs, but did not affect p46 JNK or p42/44 MAPK phosphorylation. Pretreatment with SB202190 or SP600125, specific inhibitors of p38 and JNK MAPKs, respectively, reduced ICAM-1 and VCAM-1 expression in HT29 cells dose-dependently. Adhesion of HT29 cells to FCS-coated plastic wells was inhibited dose-dependently by celecoxib, and also by SB202190 and SP600125. Celecoxib showed a pro-apoptotic effect, inducing Bax and BID but down-regulating Bcl-2. Conclusions and implications: Our findings show that celecoxib caused down-regulation of ICAM-1 and VCAM-1, affecting the adhesive properties of HT29 cells in a COX-2 independent way, inhibiting p38 and p55 MAPKs and activating a pro-apoptotic pathway. British Journal of Pharmacology (2008) 153, 870–878; doi:10.1038/sj.bjp.0707634; published online 17 December 2007

Background and purpose: Cyclooxygenase-2 (COX-2) is highly expressed during inflammation and can promote the progression of colorectal cancer. Interactions between cancer cells and vascular endothelial cells are key events in this process. Recently, the selective COX-2 inhibitor, celecoxib, was shown to inhibit expression of the adhesion molecules, ICAM-1 and VCAM-1, in the human colon cancer cell line HT29 and to inhibit adhesion of HT29 cells to FCS-coated plastic wells. Here, we evaluated the effects of celecoxib on adhesion of HT29 cells to human umbilical vein endothelial cells (HUVEC), mediated by ICAM-1 and VCAM-1, to assess further the potential protective effects of celecoxib on cancer development. Experimental approach: Celecoxib was incubated for 4 h with HT29 cells and HUVEC and adhesion was quantified by a computerized micro-imaging system. Expression analysis of ICAM-1 and VCAM-1 cell adhesion molecules was performed by western blot. Key results: Celecoxib (1 nM–10mM) inhibited, with the same potency, adhesion of HT29 cells to resting HUVEC or to HUVEC stimulated by tumour necrosis factor-a (TNF-a), mimicking inflammatory conditions. Analysis of ICAM-1 and VCAM-1 expression showed that celecoxib inhibited expression of both molecules in TNF-a-stimulated HUVEC, but not in resting HUVEC; inhibition was concentration-dependent and maximal (about 50%) at 10mM celecoxib. Conclusions and implications: In conclusion, our data show that celecoxib inhibits HT29 cell adhesion to HUVEC and expression of ICAM-1 and VCAM-1, in stimulated endothelial cells. These effects may contribute to the chemopreventive activity of celecoxib in the development of colorectal cancer. British Journal of Pharmacology (2008) 153, 1153–1161; doi:10.1038/sj.bjp.0707636; published online 17 December 2007

This study investigated the effects of the selective peroxisome proliferator-activated receptor-alpha (PPAR-alpha) agonist WY14643 on ischemia/reperfusion (I/R) injury in the rat hippocampus. Transient cerebral ischemia (30 min), followed by 1-24 h reperfusion, significantly increased the generation of reactive oxygen species, nitric oxide (NO), and lipid peroxidation end-products, as well as markedly reducing levels of the endogenous antioxidant glutathione. Reperfusion for 3-6 h led to increased expression of the proteins heme oxygenase-1 (HO-1), cyclooxygenase-2 (COX-2), inducible NO synthase (iNOS), and intercellular adhesion molecule-1 (ICAM-1). Pretreatment with WY14643 suppressed oxidative stress and expression of HO-1, iNOS, and ICAM-1, but had no effect on COX-2. These effects are due to suppression of the activation of p38 mitogen-activated protein kinase and nuclear factor-kappaB. The PPAR-alpha antagonist MK886 abolished the beneficial effects of WY14643. The levels of S100B protein, a marker of cerebral injury used in stroke trials to monitor injury, were high in the hippocampus of rats exposed to I/R, but markedly reduced by WY14643. We propose that WY14643 protects the brain against excessive oxidative stress and inflammation and may thus be useful in treating stroke.

1 Adhesion of polymorphonuclear cells (PMNs) to vascular endothelial cells (EC) is a critical step in recruitment and infiltration of leukocytes into tissues during inflammation. High doses of butyric acid have been shown to ameliorate inflammation in inflammatory bowel diseases (IBD). Cholesteryl-butyrate solid lipid nanoparticles (chol-but SLN) as prodrug are a possible delivery system for butyric acid. 2 Sodium butyrate or chol-but SLN were coincubated with human PMNs and human umbilical vein EC (HUVEC); adhesion was quantified by computerized microimaging fluorescence analysis. Both chol-but SLN and sodium butyrate displayed antiadhesive effects on FMLP- and IL-1β-stimulated cells in a concentration–response curve (10−8–10−5 M), but chol-but SLN were in all cases more active. Moreover, chol-but SLN inhibited FMLP-induced adhesion of PMNs to FCS-coated plastic wells, thus showing a direct effect on PMNs, while sodium butyrate had little effect. Confocal microscopy showed that fluorescent SLN entered PMNs and HUVEC after 10 min incubation. Chol-but SLN acted either on activated PMN or HUVEC. 3 Chol-but SLN inhibited O2−· production and myeloperoxidase release by PMNs evoked by FMLP, in a dose-dependent, but not time-dependent, manner and were more active than sodium butyrate. 4 In conclusion, in all tests chol-but SLN were more active than sodium butyrate. Thus, chol-but SLN might be a valid alternative to sodium butyrate in the anti-inflammatory therapy of ulcerative colitis, avoiding complications related to the administration of sodium butyrate. British Journal of Pharmacology (2006) 148, 648–656. doi:10.1038/sj.bjp.0706761

Substance P (SP) is a neuropeptide involved in neurogenic inflammation and an agonist for NK1, NK2, and NK3 receptors. SP induces prostaglandin (PG) production in various cell types, and these eicosanoids are responsible for numerous inflammatory and vascular effects. Cyclooxygenase (COX) are needed to convert arachidonic acid to PGs. The study evaluated the effect of SP on COX expression in human umbilical vein endothelial cells (HUVEC). COX-2 protein expression was upregulated by SP with a peak at 100 nM and at 20 h; in the same experimental conditions COX-1 protein expression was unchanged. A correlation between COX-2 expression and PGI2 and PGE2 release was detected. Dexamethasone (DEX) inhibited SP-mediated COX-2 expression. Mitogen-activated protein kinases (MAPK) p38 and p42/44 were activated by SP, whereas SB202190 and PD98059, inhibitors of these kinases, blocked COX-2 expression. 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone (DFU), an experimental selective COX-2 inhibitor, blocked SP-induced PG release. By RT–PCR and Western blot analysis, we demonstrated that NK1 and NK2 but not NK3 receptors are present on HUVEC. Selective NK1 and NK2 agonists, namely [Sar9, Met(O2)11]SP and [β-Ala8] NKA(4–10), upregulated COX-2 protein expression and PG production, whereas senktide (Suc–Asp–Phe–MePhe–Gly–Leu–Met–NH2), a selective NK3 agonist, was ineffective in this respect. The NK1 selective antagonist L703,606 ((cis)-2-(diphenylmethyl)-N-((2-iodophenyl)-methyl)-1-azabicyclo(2.2.2)octan-3-amine) and the NK2 selective antagonist SR 48,968 ((S)-N-methyl-N-(4-(4-acetylamino-4-phenylpiperidino)-2-(3,4 dichlorophenyl)butyl) benzamide) competitively antagonised SP-induced effects. The study shows HUVEC to possess functional NK1 and NK2 receptors, which mediate the ability of SP to induce expression of COX-2 in HUVEC, thus showing a previously-undetected effect of SP on endothelial cells. Keywords: Substance P, cyclooxygenase-2, HUVEC, MAPKs, NK1 receptor, NK2 receptor Introduction Substance P (SP) is a neuropeptide belonging to the tachykinin family, a group of regulatory peptides sharing the common C-terminal sequence Phe–X–Gly–Leu–Met–NH2 (Patacchini & Maggi, 1995). Other members of this family found in mammals are neurokinin A (NKA) and neurokinin B (NKB). SP is released from unmyelinated sensory nerve endings, thus evoking inflammatory peripheral effects such as vasodilatation, plasma extravasation and leukocyte activation, which are collectively referred to as ‘neurogenic inflammation' (Foreman & Jordan, 1984; Brunelleschi et al., 1991; Joos & Pauwels, 2000; Dianzani et al., 2001; Harrison & Geppetti, 2001). The effects of tachykinins are mediated by specific G-protein-coupled receptors, of which at least three subtypes have been characterised: NK1, NK2, and NK3. The existence of an NK4 receptor in man has not yet been conclusively demonstrated (Patacchini & Maggi, 1995; Page & Bell, 2002). The mammalian tachykinins display different selectivities for their receptors: SP is more selective for NK1, NKA for NK2, and NKB for NK3 (Regoli et al., 1994). Using selective SP-receptor agonists and antagonists, we recently demonstrated that SP induces PMN adhesion to human umbilical vein endothelial cells (HUVEC) and that this effect is mediated by NK1 and NK2 receptors (Dianzani et al., 2003). In the present study, the presence of SP receptors on HUVEC is investigated by RT–PCR and Western blot techniques; the results confirm that NK1 and NK2 but not NK3 receptors are present on HUVEC. Cyclooxygenase, known also as prostaglandin H2 synthase (PGHS), is the enzyme that catalyses the first two steps in the biosynthesis of prostaglandins (PGs) from arachidonic acid (Vane et al., 1998). About a decade ago, COX was shown to exist as at least two distinct isoforms, COX-1 and COX-2. COX-1 is responsible for PG and thromboxane production in gastric mucosa and platelets; COX-2 is expressed constitutively in some organs, including the kidney and the brain (Vane et al., 1998). HUVEC are known to possess both COX isoforms and to display, at Western blot analysis, variable basal levels of COX-2: either no band, or a weakly measurable band. This discrepancy may depend on cell isolation and culture procedures employed in different studies (Hirai et al., 1999; Caughey et al., 2001; Uracz et al., 2002). COX-2 induction in HUVEC has been demonstrated to occur in response to different proinflammatory cytokines, such as IL-1α and β or TNF- α (Caughey et al., 2001; Eligini et al., 2001). SP evokes the release of PGs from various cell types, including cultured spinal cord astrocytes (Koyama et al., 1999; Marriott et al., 1991), human nasal epithelial cells (Jallat-Daloz et al., 2001) and HUVEC (Alhenc-Gelas et al., 1982; Marceau et al., 1989). However, in the latter study, neither COX expression nor the underlying cell mechanisms were investigated. SP has been shown to stimulate DNA synthesis in several cell types, by activating the p38 and p42/44 mitogen-activated protein kinases (MAPK); this activation is an important signalling mechanism that participates in the development of the inflammatory process (Kyriakis & Avruch, 2001; Yang et al., 2002). MAPKs are involved in regulating PG biosynthesis, and COX-2 induction is associated with their phosphorylation (Guan et al., 1998). The study addressed the question of whether SP affects COX-2 expression in HUVEC. It investigated time- and dose-dependence of SP-evoked COX-2 expression and of PGI2 (measured as 6-keto-PGF1α) and PGE2 release from HUVEC challenged by SP. The pharmacological modulation of these events was evaluated using dexamethasone (DEX) to modulate gene expression of COX-2, and using 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone (DFU) as experimental selective COX-2 inhibitor. The involvement of p38 and p42/44 MAPKs was evaluated using two inhibitors of these kinases, SB202190 and PD98059. SP-receptors were identified on HUVEC by RT–PCR and Western blot techniques. The effects of SP were characterised in terms of receptors through NK selective agonists and antagonists.

The ability of some 2-alkyl(aryl)-4,6-dimethoxy-1,3,5-triazine derivatives to interfere with production of reactive oxygen species (ROS) by human phagocytes was evaluated in an in-vitro cell model. Superoxide anion (O2−.) production by human polymorphonuclear cells (PMNs), challenged by the chemotactic agent N-formylmethionyl-leucyl-phenylalanine (FMLP), was inhibited in a dose-dependent manner by all the compounds tested, compounds 3, 4 and 5 being statistically the most active. Adhesion of PMNs to vascular endothelial cells (ECs) is a critical step in recruitment and infiltration of leucocytes into tissues during inflammation, and the effects of 1,3,5-triazine derivatives on PMN adhesion to ECs from the human umbilical vein (HUVEC) were also investigated. Triazines were incubated with PMNs and HUVEC; adhesion was quantitated by computerized micro-imaging fluorescence analysis. The 1,3,5-triazines tested inhibited the adhesion evoked by pro-inflammatory stimuli, such as platelet activating factor (PAF), FMLP, phorbol myristate acetate (PMA), tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in a dose—response manner over the concentration range 10−9 to 10−4 m, compounds 5 and 6 being the most active. Both of these compounds inhibited PMN adhesion to HUVEC, even when endothelial or PMN stimuli were used. Indeed, when both cell populations were activated contemporarily, the anti-adhesive effect was enhanced. The study suggests that 2-aryl-4,6-dimethoxy-1,3,5-triazines deserve further evaluation as anti-inflammatory agents.

Agonists of the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) exert protective effects in several models of ischemia/reperfusion injury, but their role in stroke is less clear. The study investigates the effects of two PPAR-gamma agonists, rosiglitazone and pioglitazone, on oxidative stress and inflammatory response induced by ischemia/reperfusion in the rat hippocampus. Common carotid artery occlusion for 30 min followed by 1 h reperfusion resulted in a significant increase in the generation of reactive oxygen species, nitric oxide and the end products of lipid peroxidation as well as markedly reduced endogenous antioxidant glutathione levels and up-regulated superoxide dismutase activity. Western blot analysis showed that ischemia/reperfusion lead to an increase in cyclooxygenase-2 (COX-2) expression, as well activating p38 and p42/44 mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB). Pre-treatment with either rosiglitazone or pioglitazone significantly reduced oxidative stress, COX-2 protein expression and activation of MAPKs and NF-kappaB. Taken together, the results provide convincing evidence that PPAR-gamma agonists exert protective effects in a rat model of mild forebrain ischemia/reperfusion injury by inhibiting oxidative stress and excessive inflammatory response.

GAS6, the product of a growth arrest specific (GAS) gene, is the ligand of the tyrosine kinase receptor Axl. GAS6 and Axl are both expressed in endothelial cells, where they are involved in many processes such as leukocyte transmigration through capillaries and neointima formation in injured vessels. Here, we show that Axl stimulation by GAS6 results in inhibition of the ligand-dependent activation of vascular endothelial growth factor (VEGF) receptor 2 and the consequent activation of an angiogenic program in vascular endothelial cells. GAS6 inhibits chemotaxis of endothelial cells stimulated by VEGF-A isoforms, but not that triggered by fibroblast growth factor-2 or hepatocyte growth factor. Furthermore, it inhibits endothelial cell morphogenesis on Matrigel and VEGF-A–dependent vascularization of chick chorion allantoid membrane. GAS6 activates the tyrosine phosphatase SHP-2 (SH2 domain-containing tyrosine phosphatase 2), which is instrumental in the negative feedback exerted by Axl on VEGF-A activities. A dominant-negative SHP-2 mutant, in which Cys 459 is substituted by Ser, reverted the effect of GAS6 on stimulation of VEGF receptor 2 and endothelial chemotaxis triggered by VEGF-A. These studies provide the first demonstration of a cross talk between Axl and VEGF receptor 2 and add new information on the regulation of VEGF-A activities during tissue vascularization.

Among the gynecological malignancies, ovarian cancer is the leading cause of mortality in developed countries. Treatment of ovarian cancer is based on surgery integrated with chemotherapy. Platinum-based drugs (cisplatin and carboplatin) comprise the core of first-line chemotherapy for patients with advanced ovarian cancer. Platinum-resistant ovarian cancer can be treated with cytotoxic chemotherapeutics such as paclitaxel, topotecan, PEGylated liposomal doxorubicin, or gemcitabine, but many patients eventually relapse on treatment. Targeted therapies based on agents specifically directed to overexpressed receptors, or to selected molecular targets, may be the future of clinical treatment. In this regard, overexpression of folate receptor-α on the surface of almost all epithelial ovarian cancers makes this receptor an excellent "tumor-associated antigen". With appropriate use of spacers/linkers, folate-targeted drugs can be distributed within the body, where they preferentially bind to ovarian cancer cells and are released inside their target cells. Here they can exert their desired cytotoxic function. Based on this strategy, 12 years after it was first described, a folate-targeted vinblastine derivative has now reached Phase III clinical trials in ovarian cancer. This review examines the importance of folate targeting, the state of the art of a vinblastine folate-targeted agent (vintafolide) for treating platinum-resistant ovarian cancer, and its diagnostic companion (etarfolatide) as a prognostic agent. Etarfolatide is a valuable noninvasive diagnostic imaging agent with which to select ovarian cancer patient populations that may benefit from this specific targeted therapy.

Acquired immunodeficiency syndrome (AIDS)-related non-Hodgkin's lymphomas (AIDS-NHLs) consistently derive from B cells, are histologically heterogeneous, and are associated with distinct molecular pathways depending upon histology. Recently, it has been proposed that inactivating mutations of the bax death agonist may contribute to the pathogenesis of human tumors. In particular, among B-cell malignancies, BAX mutations have been detected at a certain frequency in Burkitt lymphomas. This study is aimed at defining the status of the BAX gene throughout the clinicopathologic spectrum of AIDS-NHL (n = 54), including AIDS-related Burkitt lymphoma (n = 14), AIDS-related Burkitt-like lymphoma (n = 8), AIDS-related diffuse large cell lymphoma (n = 15), AIDS-related primary central nervous system lymphoma (n = 6), and AIDS-related primary effusion lymphoma (n = 11). All 6 BAX exons and flanking sequences were subjected to mutational analysis by polymerase chain reaction-single strand conformation polymorphism followed by DNA direct sequencing of positive cases. Mutations of BAX among AIDS-NHL were restricted to a cell line of AIDS-related primary effusion lymphoma, which harbored a frameshift mutation causing the introduction of a proximal stop codon. All other AIDS-NHL displayed wild-type BAX alleles. In order to investigate whether BAX inactivation in AIDS-NHL may occur through mechanisms other than gene mutation, bax protein expression was investigated by Western blot analysis or immunohistochemistry in selected cases. All AIDS-NHL analyzed expressed normal bax proteins. Overall, this study indicates that deregulation of apoptotic control in AIDS-NHL is not caused by BAX alterations. Genes Chromosomes Cancer 27:177-182, 2000.

Primary effusion lymphoma (PEL) harbors consistent infection by human herpesvirus-8, preferentially develops in immunodeficient patients and selectively localizes to the serous body cavities. Histogenetic analysis has suggested that PEL originates from post-germinal center, pre-terminally differentiated B cells sharing phenotypic features with plasma cells. Here we have investigated the expression status and functional integrity of the Met tyrosine kinase receptor and of its ligand hepatocyte growth factor (HGF). Thirteen PEL (nine cell lines and four primary specimens) were analyzed for Met and HGF expression and function by multiple assays. For comparison, a panel of 34 high grade B cell non-Hodgkin lymphomas (NHL) other than PEL was also investigated. Co-expression of Met and HGF was found in all PEL analyzed, whereas it was restricted to 1/34 B cell NHL other than PEL (P < 0.001; chi2 test). The Met protein expressed by PEL displays biochemical characteristics typical of Met expressed by other cell types and is capable of tyrosine autophosphorylation. By using a combination of immunological and biological assays, production and secretion of a functional HGF species was identified in all PEL cell lines analyzed. HGF stimulation of PEL cells rapidly induces Met tyrosine phosphorylation, demonstrating the functional integrity of the Met/HGF loop. Because of the well known mitogenic and motogenic properties of Met/HGF interactions, these data may bear implications for PEL growth and dissemination. Among B cell neoplasms, Met/HGF co-expression selectively clusters with PEL and, as demonstrated by previous studies, with multiple myeloma plasma cells, thus reinforcing the notion that PEL displays biologic similarities with tumors derived from late stages of B cell differentiation.

Recent large sequencing efforts of the human cancer genome have unveiled a highly heterogeneous pattern of mutated genes even among tumors of the same tis-sue origin. In the majority of cases, the function of these mutated alleles is not well characterized, and future studies will increasingly have to rely on cell-based models to assess the impact of mutations in individual genes on complex molecular pathways and cell functions.Two main approaches can be used to assess the phenotype of cancer-associated mutations in cell models. In the first, the cDNA corresponding to putative onco-genic alleles are introduced into cells by transfection or viral transduction. Similarly, plasmids or viral vectors can be employed to deliver inhibitory RNAs to tumor suppressor genes and assess the functional effects of gene inactivation by downregulation. However, ectopic expres-sion methods are often hampered by arti-ficially high gene expression levels; while the use of siRNA can suffer from incom-plete repression of targeted alleles and potential off-target effects.In the second approach, mutations are introduced into the genomes of human normal or tumor cells using gene-tar-geting methods. Although more labori-ous than overexpression-based methods, genetic manipulation of human somatic cells has proven valuable for a variety of purposes: to express oncogenic alleles from their endogenous promoters

Personalized cancer medicine is based on the concept that targeted therapies are effective on subsets of patients whose tumors carry specific molecular alterations. Several mammalian target of rapamycin (mTOR) inhibitors are in preclinical or clinical trials for cancers, but the molecular basis of sensitivity or resistance to these inhibitors among patients is largely unknown. Here we have identified oncogenic variants of phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) and KRAS as determinants of response to the mTOR inhibitor everolimus. Human cancer cells carrying alterations in the PI3K pathway were responsive to everolimus, both in vitro and in vivo, except when KRAS mutations occurred concomitantly or were exogenously introduced. In human cancer cells with mutations in both PIK3CA and KRAS, genetic ablation of mutant KRAS reinstated response to the drug. Consistent with these data, PIK3CA mutant cells, but not KRAS mutant cells, displayed everolimus-sensitive translation. Importantly, in a cohort of metastatic cancer patients, the presence of oncogenic KRAS mutations was associated with lack of benefit after everolimus therapy. Thus, our results demonstrate that alterations in the KRAS and PIK3CA genes may represent biomarkers to optimize treatment of patients with mTOR inhibitors.

Mandarin translation of abstract Background and purpose: We investigated the ability of natural and synthetic selective NK receptors agonists and antagonists to modulate cyclooxygenase-2 (COX-2) expression in human polymorphonuclear leucocytes (PMNs). Experimental approach: The presence of all three tachykinin in PMNs was assessed by Western blot and PCR techniques. Natural and synthetic ligands selective for the tachykinin receptors were used to modulate COX-2 protein (measured with Western blotting) and activity [as prostaglandin E2 (PGE2) output]. Effects of substance P (SP) on phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) activation were studied to analyse the signalling pathway involved in COX-2 up-regulation mediated by SP. Key results: Stimulation of NK receptors with the natural ligands SP, neurokinin A (NKA) and neurokinin B, in the pmol·L−1-µmol·L−1 concentration range, modulated COX-2 expression and PGE2 release in a concentration- and time-dependent manner. Experiments with synthetic selective agonists [Sar9, Met(O2)11]SP, [β-Ala8] NKA(4-10), senktide or selective antagonists L703,606, SR48,968 or SR142801, confirmed that COX-2 up-regulation was mediated by NK receptors. We found that mainly p38, p42 and p46 MAPKs were phosphorylated by SP and SB202190, PD98059 and SP600125, which are selective inhibitors of these kinases, blocked SP-induced COX-2 expression. SP also induced nuclear translocation of NF-κB concentration-dependently, with a maximum effect at 1 nmol·L−1. Conclusions and implications: Human PMNs possess functional NK1, NK2 and NK3 receptors, which mediate the induction of COX-2 expression and NF-κB activation by SP. Mandarin translation of abstract

The serine/threonine glycogen synthase kinase 3beta (GSK-3beta) is abundant in the central nervous system, particularly in the hippocampus, and plays a pivotal role in the pathophysiology of a number of diseases, including neurodegeneration. This study was designed to investigate the effects of GSK-3beta inhibition against I/R injury in the rat hippocampus. Transient cerebral ischemia (30 min) followed by 1 h of reperfusion significantly increased generation of reactive oxygen species and modulated superoxide dismutase activity; 24 h of reperfusion evoked apoptosis (determined as mitochondrial cytochrome c release and Bcl-2 and caspase-9 expression), resulted in high plasma levels of TNF-alpha and increased expression of cyclooxygenase-2, inducible nitric oxide synthase, and intercellular adhesion molecule-1. The selective GSK-3beta inhibitor, 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), was administered before and after ischemia or during reperfusion alone to assess its potential as prophylactic or therapeutic strategy. Prophylactic or therapeutic administration of TDZD-8 caused the phosphorylation (Ser(9)) and hence inactivation of GSK-3beta. Infarct volume and levels of S100B protein, a marker of cerebral injury, were reduced by TDZD-8. This was associated with a significant reduction in markers of oxidative stress, apoptosis, and the inflammatory response resulting from cerebral I/R. These beneficial effects were associated with a reduction of I/R-induced activation of the mitogen-activated protein kinases JNK1/2 and p38 and nuclear factor-kappaB. The present study demonstrates that TDZD-8 protects the brain against I/R injury by inhibiting GSK-3beta activity. Collectively, our data may contribute to focus the role of GSK-3beta in cerebral I/R.

OBJECTIVE Sphingosylphosphorylcholine (SPC) has been reported to activate a variety of G-protein coupled receptors, including S1P(1-5), G2A, GPR4, and OGR1 (GPR68). Interestingly, other structurally related lysophospholipid agonists of these receptors have been shown to exhibit immunomodulatory properties both in vitro and in vivo. These include prevention of tumor necrosis factor-alpha-induced monocyte adhesion to aortic endothelium in mice (sphingosine-1-phosphate via S1P(1-5) receptors) and reduction of organ injury and/or mortality in animal models of sepsis and endotoxemia (lysophosphatidylcholine via G2A). Here, we investigate the effects of SPC on the organ injury/dysfunction caused by systemic administration of lipopolysaccharide and the mechanisms underlying the observed effects of SPC. DESIGN Prospective, randomized study. SETTING University-based research laboratory. SUBJECTS Sixty-one anesthetized male Wistar rats. INTERVENTIONS Rats received either SPC (10 mg/kg intravenously) or vehicle (phosphate-buffered saline 1 mL/kg intravenously) 15 mins before or 15 mins after induction of endotoxemia with lipopolysaccharide (6 mg/kg intravenously). MEASUREMENTS AND MAIN RESULTS Treatment with SPC significantly reduced the organ/dysfunction injury caused by lipopolysaccharide. SPC pretreatment significantly reduced the circulating levels of interleukin-1beta and interleukin-6, the expression of CD11b (ligand for intercellular adhesion molecule-1) on circulating polymorphonuclear cells, the expression of proteins of intercellular adhesion molecule-1 (Western blot and immunohistochemistry), cyclooxygenase-2 and nuclear translocation of nuclear factor-kappaB (Western blot analysis), and inducible nitric oxide synthase (immunohistochemistry) as well as the lung injury caused by endotoxemia in the rat. CONCLUSIONS SPC reduced the organ injury/dysfunction caused by endotoxin in the rat. These beneficial effects of SPC are associated with potent anti-inflammatory effects.

The generation of endogenous hydrogen sulfide may either limit or contribute to the degree of tissue injury caused by ischemia/reperfusion. A total of 74 male Wistar rats were used to investigate the effects of endogenous and exogenous hydrogen sulfide in renal ischemia/reperfusion. Administration of the irreversible cystathionine gamma-lyase (CSE) inhibitor, dL-propargylglycine, prevented the recovery of renal function after 45 min ischemia and 72 h reperfusion. The hydrogen sulfide donor sodium hydrosulfide attenuated the (renal, tubular, and glomerular) dysfunction and injury caused by 45 min ischemia and 6 h reperfusion. Western blot analysis of kidneys taken at 30 min reperfusion showed that sodium hydrosulfide significantly attenuated phosphorylation of mitogen-activated protein kinases (p-38, c-JUN N-terminal protein kinase 1/2, and extracellular signal-regulated kinase 1/2) and activation of nuclear factor-kappaB. At 6 h reperfusion, sodium hydrosulfide significantly attenuated the histological score for acute tubular necrosis, the activation of caspase-3 and Bid, the decline in the expression of anti-apoptotic Bcl-2, and the expression of nuclear factor-kappaB-dependent proteins (inducible nitric oxide synthase, cyclo-oxygenase-2, and intercellular adhesion molecule-1). These findings suggest that (1) the synthesis of endogenous hydrogen sulfide by CSE is essential to protect the kidney against ischemia/reperfusion injury and dysfunction and aids in the recovery of renal function following ischemia/reperfusion, (2) hydrogen sulfide generated by sodium hydrosulfide reduces ischemia/reperfusion injury and dysfunction, and morphological changes of the kidney, and (3) the observed protective effects of hydrogen sulfide are due to both anti-apoptotic and anti-inflammatory effects.

Mutations in oncogenes and tumor suppressor genes are responsible for tumorigenesis and represent favored therapeutic targets in oncology. We exploited homologous recombination to knock-in individual cancer mutations in the genome of nontransformed human cells. Sequential introduction of multiple mutations was also achieved, demonstrating the potential of this strategy to construct tumor progression models. Knock-in cells displayed allele-specific activation of signaling pathways and mutation-specific phenotypes different from those obtainable by ectopic oncogene expression. Profiling of a library of pharmacological agents on the mutated cells showed striking sensitivity or resistance phenotypes to pathway-targeted drugs, often matching those of tumor cells carrying equivalent cancer mutations. Thus, knock-in of single or multiple cancer alleles provides a pharmacogenomic platform for the rational design of targeted therapies.

Background[1, 2, 4] triazolo [4, 3-a][1, 8]naphthyridine derivatives (including NF161 and NF177) were tested for anti-inflammatory, analgesic and antipyretic properties and for their effects on spontaneous locomotor activity in mice and acute gastrolesivity in rats. Both NF161 and NF177 appeared to be anti-inflammatory and analgesic agents without toxic effects or acute gastrolesivity, but NF161 showed stronger anti-inflammatory activity, whereas NF177 was more active as analgesic.MethodsAn EIA kit was used to investigate the ability of NF161 and NF177 to affect prostaglandin E2(PGE2) and prostacyclin (PGI2) production by human umbilical vascular endothelial cells (HUVEC).The compounds' effects on the production of reactive oxygen species (ROS) by human polymorphonuclear cells (PMNs) were studied in anin vitrocell model, evaluating inhibition of superoxide anion (O2-.) production induced byN-formylmethionyl-leucyl-phenylalanine (FMLP). Their effects on PMN adhesion to HUVEC were also investigated; they were incubated with PMNs and endothelial cells (EC) and challenged by stimuli including Platelet Activating Factor (PAF), FMLP, Phorbol Myristate Acetate (PMA), Tumor Necrosis Factor-α (TNF-α) and Interleukin-1β (IL-1β). Adhesion was quantitated by computerized micro-imaging fluorescence analysis.ResultsNeither compounds modified PGE2or PGI2production induced by IL-1α.O2-.production and myeloperoxidase release from PMNs stimulated by FMLP was inhibited in a dose- but not time-dependent manner by both [1, 8]naphthyridine derivatives, NF161 being statistically more active than NF177 (P < 0.01).The compounds inhibited adhesion evoked by the pro-inflammatory stimuli PAF, FMLP, TNF-α and IL-1β in a concentration-dependent manner in the 10-6–10-4M range, being more active when PAF was used as stimulus and inactive when cells were challenged by PMA. Both compounds acted both on PMN and HUVEC.ConclusionConsidering the interesting anti-inflammatory effects of these compounds inin vivomodels and the absence of acute gastrolesivity, the study improved knowledge of anti-inflammatory properties of NF161 and NF177, also demonstrating their potentialin vitro, through inhibition of O2-.production, myeloperoxidase release and PMN adhesion to HUVEC. Negative results on PG production suggest a cyclooxygenase (COX)-independent mechanism.

In Fura-2/loaded ECV304 endothelial cells cyclovirobuxine D promoted a transient increase in cytosolic free Ca2+ originating from both an intracellular pool sensitive to the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin and the extracellular space. The intracellular pool was apparently different from that mobilized by other agents acting through IP3 generation. The integrity of the plasma membrane was an absolute requirement. In cells treated with digitonin, cyclovirobuxine D did not promote any Ca2+ release from the intracellular stores even at high concentrations and in the absence or presence of thapsigargin or sodium azide, the inhibitors of the endoplasmic reticular or mithocondrial Ca2+ uptake. Furthermore, cyclovirobuxine D was effective in halting the persistent increase in cytosolic Ca2+ caused by thapsigargin, inhibiting the operation of the “capacitative” Ca2+ membrane channels as demonstrated by the decrease in the extent of both Ca2+-overshoot and Mn2+ influx. Additional effects of cyclovirobuxine D included a depolarization of plasma membrane apparently related to an enhanced influx of Na+ from the extracellular space. The results obtained indicate that cyclovirobuxine D markedly affects intracellular Ca2+ homeostasis in ECV304 endothelial cells by both promoting a discharge of intracellular pools and by interfering with the operation of store-dependent channels via plasma membrane depolarization.

Clonal hematopoiesis is the hallmark of myelodysplastic syndromes, but the role played by pluripotent stem cells and progenitor cells in these disorders remains unclear.Eight female patients with myelodysplastic syndrome were studied. X-chromosome inactivation patterns were analyzed in peripheral blood granulocytes, T-lymphocytes, single colonies originating from bone marrow progenitors and pluripotent stem cells, using the human androgen receptor locus polymorphism assay.Granulocytes and progenitor cells were monoclonal in 7/8 cases. Immature stem cells showed a non-clonal pattern of X-inactivation and were detectable at diagnosis in the presence of clonal hematopoiesis. T-lymphocyte clonality was heterogeneous.In myelodysplastic syndromes, hematopoiesis may be dominated by a neoplastic clone by virtue of its biological advantage over a residual polyclonal, probably still normal, population of immature stem cells still able to grow in vitro.

Background: Primary effusion lymphoma (PEL) associates with HHV-8 infection, preferentially develops in immunodeficient patients and grows in the serous body cavities. PEL derives from post-germinal center, pre-terminally differentiated B-cells. The pathogenesis of PEL is unclear and the sole identified genetic lesions are human herpesvirus type-8 (HHV-8) infection in all cases and EBV infection in 70% of cases. Epstein–Barr virus (EBV) infection in PEL displays a latency I phenotype. Objectives: To clarify the pathogenesis and histogenesis of PEL by investigating (1) the lymphoma karyotype; (2) the expression status of the Met tyrosine kinase receptor and of its ligand hepatocyte growth factor (HGF); (3) the molecular profile of EBV, with particular focus on mutations of EBNA-1 genes, which are thought to affect viral tumorigenicity in EBV-infected neoplasms displaying the latency I phenotype. Study design: Twenty-four PEL (nine cell lines and 15 primary specimens) formed the basis of the study. Karyotypes were investigated by conventional cytogenetics and fluorescent in situ hybridization (FISH) in selected cases. The expression status of Met and HGF was defined by multiple techniques, including RT-PCR, FACS analysis, immunocytochemistry, Western blot studies and ELISA. The molecular profile of EBNA-1 genes of EBV were investigated by DNA direct sequencing. Results: Trisomy 7, trisomy 12 and breaks at 1q21–q25 are recurrently associated with PEL. PEL consistently co-express Met and HGF both at the mRNA and protein level. Among aggressive B-cell lymphomas, Met/HGF co-expression appears to be relatively specific for PEL. The EBNA-1 gene of EBV displays a high degree of genetic heterogeneity in PEL, with no preferential association with one specific variant. Conclusions: PEL associates with recurrent chromosomal alterations, suggesting that viral infection is not sufficient for tumor development and that lesions of cellular genes may be required. The expression of Met/HGF by PEL cells may bear implications for the lymphoma proliferation and growth pattern, since Met/HGF interactions influence cell mitogenesis and motogenesis. EBV infection in PEL displays a latency I phenotype and fails to associate with specific EBNA-1 variants, suggesting that the role of EBV in PEL is not mediated by the major transforming pathways currently known in EBV positive lymphomas.

Hematopoietic growth factors (HGFs) sustain the survival, proliferation and differentiation of hematopoietic stem cells and some functions of mature blood cells. In man several HGFs have been characterised and cloned so far, and this has allowed investigators to confer the rationale for the clinical application of these molecules in hematology and oncology. In particular G-CSF and GM-CSF are currently utilised to abrogate the hematological toxicity of chemotherapy for standard and dose-intensified therapy, neutropenia following bone marrow and peripheral blood stem cell transplantation. Moreover there has recently been great interest in the ex vivo expansion of hematopoietic stem and progenitor cells for a variety of applications, such as in vitro tumor cell purging or for reducing the volume of blood processed by the leukapheresis. Several combinations of HGFs have been described to sustain the ex vivo survival and proliferation of these cells disclosing new opportunities in the field of stem cells transplants.

A main limitation of therapies that selectively target kinase signaling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain of EGFR, is effective in a subset of KRAS wild type metastatic colorectal cancers. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood. Here, we show for the first time that molecular alterations (in most instances point mutations) of KRAS are causally associated with the onset of acquired resistance to anti-EGFR treatment in colorectal cancers. Expression of mutant KRAS under the control of its endogenous gene promoter was sufficient to confer cetuximab resistance but resistant cells remained sensitive to combinatorial inhibition of EGFR and MEK (Figure 1). Analysis of metastases from patients who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60 % (6/10) of the cases (Table 1). KRAS mutant alleles were detectable in the blood of cetuximab treated patients as early as 10 months prior to radiographic documentation of disease progression. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancers, indicate that the emergence of KRAS mutant clones can be detected non-invasively months prior to radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance.

Ras mutations are present in approximately 49% of human colorectal cancers, with mutations in K-Ras G12 and G13 being the most common. Given their high prevalence, understanding how Ras mutations impact response to anticancer agents is crucial to the successful development of novel agents. Recent evidence suggests that not all variants of K-Ras respond in the same way to targeted agents, where some patients with G13D mutations respond better to Cetuximab compared to patients expressing G12V (De Roock, JAMA 2011). Therefore there is a need for better in vitro models to facilitate the characterisation of inhibitor selectivity and efficacy in K-Ras mutant cell systems. We created suites of isogenic X-MAN SW48 and LIM1215 colorectal cancer cells, each of which harbour one of 7 different K-Ras G12 or G13 mutations at their endogenous loci introduced via AAV-mediated homologous recombination. This system allows use of endogenous promoters and has enabled us to create a panel of human cell lines in which the only difference between the cells is the point mutation of interest. This more accurately recapitulates the genomic architecture in human tumors and provides an excellent platform for addressing the impact of K-Ras mutations on responses to therapeutic agents. We selected over 30 targeted agents and cytotoxic drugs based on their clinical relevance to colorectal cancer. We profiled the anti-proliferative activity of each of these across the suite of K-Ras variants in SW48 cells. Strikingly, we found a consistent pattern of resistance towards 5 chemically distinct MEK1/2 inhibitors across the 7 K-Ras variants compared to wild-type cells. These results were confirmed in a second suite of K-Ras mutant lines generated in LIM1215 cells, where clear resistance to MEK inhibitors was observed. To investigate the mechanism of this resistance to MEK inhibitors, we combined the anti-EGFR monoclonal antibody Cetuximab with the MEK inhibitor AZD6244, both compounds being ineffective as single agents in the K-Ras G12D and G12C mutant SW48 cells. Concentrations of Cetuximab as low as 0.01μg/ml re-sensitised previously resistant K-Ras mutant cells to the effects of AZD6244. Given that this dose of Cetuximab was ineffective alone, this result suggests that upregulation of the EGFR axis may be a potential mechanism in compensating for MEK inhibition, and these studies are on-going. In conclusion, we have created two suites of isogenic colorectal cancer cell lines each expressing a different K-Ras G12 or G13 mutation and demonstrated that K-Ras mutations impart resistance to clinical MEK inhibitors. Despite screening over 30 chemotherapeutics covering an array of targets, we did not identify any other significant differences in sensitivity between K-Ras wild-type and mutant cells, demonstrating the mechanistic relevance of this model to MEK inhibitors. Our data highlight the advantages of using this system in profiling molecularly targeted agents in order to identify potential mechanisms of resistance and in rationally identifying combinations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A70.

The mutational landscape of cancer genomes displays a complex combination of genetic lesions affecting oncogenes and tumor suppressor genes. The construction of cellular models closely recapitulating cancer-linked genetic alterations is a prerequisite to understand their role in tumor progression and to identify genotype- specific pharmacological responses. To recapitulate point mutations affecting oncogenes we used homologous recombination to ‘knock-in’ specific nucleotide changes in the genome of human cells. We focused on the following alleles: EGFR (delE746-A750), KRAS (G13D), BRAF (V600E) and PIK3CA (E545K and H1047R), that are found in multiple human cancers. As recipient cells, a human non transformed epithelial cell line of breast origin (hTERT-HME1) has been employed. This cell line has been immortalized by hTERT and harbors a mutation on TP53 gene, a genetic event frequently observed in naturally occurring human tumors. To recapitulate the inactivation of additional tumor suppressor genes in our cellular models, we exploited shRNAs to permanently knock-down the expression of PTEN and RB1 genes, as they are frequently down regulated in human tumors. Knock-down of PTEN and RB1 was performed both in the parental hTERT-HME1 cells and in the ‘knock-in’ models, thus allowing us to combine tumor suppressor genes inactivation with mutational activation of specific oncogenes. The resulting cellular models are hereafter refereed to as combinatorial ‘matrix’. To assess how specific combinations of genetic lesions affect the tumorigenic properties of not transformed cells, we measured the ability of the ‘matrix’ to grow in soft agar. At the same time we injected our cellular models in the cleared, humanized mammary fat pad of immunocompromised mice. For the latter approach, we focused on genotypes recapitulating mutations found in breast tumors, such as PTEN or RB1 inactivation and PIK3CA or KRAS mutations, alone or in combination. In vitro and in vivo results showed that those combinations of genetic events are not sufficient to transform the normal hTERT-HME1 cells. However, preliminary data indicates that few genotypes of the ‘matrix’ acquired the ability to colonize the murine fat pads. With the rational of unveiling new pharmaco-genetic interactions involving one or multiple cancer mutations, the “matrix” was used to screen a panel of compounds used for cancer therapy, or for which an anti-proliferative activity was previously reported. This approach led to the identification of a subset of genotype-drug interactions thus underscoring how the multifactorial genomic milieu of cancer cells impact the response to therapies. Grant support: This work is part of the EU FP7 grant COLTHERES Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 230. doi:10.1158/1538-7445.AM2011-230

a Department of Anatomy, Pharmacology, and Forensic Medicine, University of Turin, 10125 Turin, Italy b Department of Experimental Medicine and Oncology, University of Turin, 10125 Turin, Italy c Centre for Experimental Medicine, Nephrology and Critical Care, William Harvey Research Institute, St. Bartholomew’s and The Royal London School of Medicine and Dentistry, Queen Mary University of London, London, UK