TLN-4601 suppresses growth and induces apoptosis of pancreatic carcinoma cells through inhibition of Ras-ERK MAPK signaling

Abstract Background TLN-4601 is a structurally novel farnesylated dibenzodiazepinone discovered using Thallion's proprietary DECIPHER® technology, a genomics and bioinformatics platform that predicts the chemical structures of secondary metabolites based on gene sequences obtained by scanning bacterial genomes. Our recent studies suggest that TLN-4601 inhibits the Ras-ERK MAPK pathway post Ras prenylation and prior to MEK activation. The Ras-ERK MAPK signaling pathway is a well-validated oncogenic cascade based on its central role in regulating the growth and survival of cells from a broad spectrum of human tumors. Furthermore, RAS isoforms are the most frequently mutated oncogenes, occurring in approximately 30% of all human cancers, and KRAS is the most commonly mutated RAS gene, with a greater than 90% incidence of mutation in pancreatic cancer. Results To evaluate whether TLN-4601 interferes with K-Ras signaling, we utilized human pancreatic epithelial cells and demonstrate that TLN-4601 treatment resulted in a dose- and time-dependent inhibition of Ras-ERK MAPK signaling. The compound also reduced Ras-GTP levels and induced apoptosis. Finally, treatment of MIA PaCa-2 tumor-bearing mice with TLN-4601 resulted in antitumor activity and decreased tumor Raf-1 protein levels. Conclusion These data, together with phase I/II clinical data showing tolerability of TLN-4601, support conducting a clinical trial in advanced pancreatic cancer patients

Synergistic activity of troxacitabine (Troxatyl™) and gemcitabine in pancreatic cancer

<p>Abstract</p> <p>Background</p> <p>Gemcitabine, a deoxycytidine nucleoside analog, is the current standard chemotherapy used as first-line treatment for patients with locally advanced or metastatic cancer of the pancreas, and extends life survival by 5.7 months. Advanced pancreatic cancer thus remains a highly unmet medical need and new therapeutic agents are required for this patient population. Troxacitabine (Troxatyl™) is the first unnatural L-nucleoside analog to show potent preclinical antitumor activity and is currently under clinical investigation. Troxacitabine was recently evaluated as a first-line therapy in 54 patients with advanced adenocarcinoma of the pancreas and gave comparable overall results to those reported with gemcitabine in recently published randomized trials.</p> <p>Methods</p> <p>The human pancreatic adenocarcinoma cell lines, AsPC-1, Capan-2, MIA PaCa-2 and Panc-1, were exposed to troxacitabine or gemcitabine alone or in combination, for 72 h, and the effects on cell growth were determined by electronic particle counting. Synergistic efficacy was determined by the isobologram and combination-index methods of Chou and Talalay. Mechanistic studies addressed incorporation of troxacitabine into DNA and intracellular levels of troxacitabine and gemcitabine metabolites. For <it>in vivo </it>studies, we evaluated the effect of both drugs, alone and in combination, on the growth of established human pancreatic (AsPC-1) tumors implanted subcutaneously in nude mice. Statistical analysis was calculated by a one-way ANOVA with Dunnett as a post-test and the two-tailed unpaired <it>t </it>test using GraphPad prism software.</p> <p>Results</p> <p>Synergy, evaluated using the CalcuSyn Software, was observed in all four cell-lines at multiple drug concentrations resulting in combination indices under 0.7 at Fa of 0.5 (50% reduction of cell growth). The effects of drug exposures on troxacitabine and gemcitabine nucleotide pools were analyzed, and although gemcitabine reduced phosphorylation of troxacitabine when cells were exposed at equal drug concentrations, there was no effect on phosphorylated pools at drug combinations that were synergistic. The amount of troxacitabine incorporated into DNA was also not affected by the presence of gemcitabine. <it>In vivo </it>testing against a human pancreatic (AsPC-1) xenograft mouse tumor model indicated that both drugs were more than additive at well-tolerated doses and schedule. The biological basis for this synergy is unclear as we did not observe changes in apoptosis, DNA repair, troxacitabine incorporation into DNA or troxacitabine metabolism in the presence of gemcitabine.</p> <p>Conclusion</p> <p>These data, together with phase I clinical data showing tolerability of both agents when combined, suggest combination therapy with troxacitabine and gemcitabine warrants further evaluation in advanced pancreatic cancer patients.</p

Promotion of growth and differentiation of rat ductular oval cells in primary culture

Oval cells emerging in rat liver at the early period of 3-methyl-4- dimethylaminoazobenzene treatment constitute a mixed epithelial cell compartment with respect to a-fetoprotein (AFP) and cytokeratin differ ential expression, and include a subpopulation which exhibits a phenotype intermediate between ductular cells and hepatocytes (Germain et «/., Cancer Res., 45:673-681,1985). In the present study we have examined the developmental potential of ductular oval cells in primary culture and after in vivo transfer. The use of monoclonal and poh chinai antibodies directed against cytokeratins of M, 39,000 (CK39), 52,000 (CK52), and 55,000 (CK55) and vimentin, and also monoclonal antibodies against exposed surface components of oval cells (BL)S,) and normal hepatocytes (I IFSft) allowed us to establish the ductular phenotype of the oval cells. A highly enriched preparation of oval cells was obtained by perfusion/ digestion of the liver with collagenase, treatment of the cell suspension with trypsin and DNase, selective removal of hepatocytes by panning using the anli-UKS,, antibody, and cell separation by isopyknic centrifugation in a Percoli gradient. The procedure yielded about 8 x IO1 cells, of which 95% expressed CK39, CK52, and BDS, 84% f-glutamyl transpeptidase, and 5% albumin and AFP. The primary response of cultured oval cells to various combinations of growth and differentiation promoting factors was evaluated with respect to their capacity to initiate DNA synthesis as measured by [3H)thymidine labeling from day 1 to 3, and/or to produce albumin and AFP and express tyrosine aminotransferase. Culture in the presence of either serum or clot blood extract resulted in a low proliferative activity with less than 5% of the nuclei being labeled. Over a 5-day period, fusion of a large portion of the oval cells led to multinucleated cells. When the cells were cultured in the presence of an elaborate combination of supplements [minimum essential medium containing 1 HIMpyruvate, 0.2 HIMaspartate, 0.2 HIMserine, 1 DIMtyrosine, 1 mM proline, 1 IHMphenylalanine and supplemented with 20% clot blood extract, 10 ng/ml oxidized bile acids, 17 /IM bilirubin, 10 ng/ml cholera toxin, l /IM dexamethasone, 2.5 «ig/mlinsulin, 50 HIMftmercaptoetbanol, and 5 «ig/mltransferrin (medium MX)|, the labeling index increased to around 30% and the level of cell fusion greatly decreased. The addition of dimethyl sulfoxide further enhanced the initiation of DNA synthesis, while sodium butyrate acted as an inhibitor. Cell morphology varied depending on the combinations of factors used. Most of the oval cells still expressed the three cytokeratins, HI)S7, 7- glutamyl transpeptidase, and some vimentin after 5 days in culture. Culture in medium MX in the absence of sodium butyrate resulted in the emergence of a small subpopulation of BI)S7 negative/T-glutamyl trans peptidase positive fusiform cells, which by day 5 constituted a distinct epithelial cell population that expressed CK39 and vimentin and which could be subcultured. The addition of dimethyl sulfoxide or sodium butyrate at day 1 in the presence of dexamethasone differentially modified the phenotype of the ductular oval cells cultured in growth factorsupplemented medium. Of major interest was the finding that the pres ence of sodium butyrate and dexamethasone led to a massive production of albumin over a 3-day period and a progressive induction of tyrosine aminotransferase activity. Fourteen days after the injection of freshly isolated oval cells into the fat pads of syngeneic rats, the cells were arranged as clusters similar to bile ductular structures. These findings indicate that oval cells isolated from 3-methyl-4-dimethylaminoazobenzene-treated rat liver which exhibit the phenotypic characteristics of bile ductular epithelial cells after in vivo transfer, produce albumin, cease AFP production, and express tyrosine aminotransferase activity in pri mary mass culture, properties of facultative hepatocytic precursors

Cell of origin of distinct cultured rat liver epithelial cells, as typed by cytokeratin and surface component selective expression

L'origine intra-tissulaire des cellules épithéliales non-parenchymateuses émergeant des cultures de cellules hépatiques demeure inconnue. Des cultures d'hépatocytes et de cellules non-parenchymateuses de foie de rat obtenues par des méthodes sélectives de dispersion du tissu ont été analysées à l'aide d'anticorps monoclonaux réagissant avec les cytokératines et la vimentine de foie de rat, d'anticorps polyvalents dirgés contre les cytokératines de sabots de vache et la vimentine de lentilles de porc, et d'anticorps monoclonaux dirigés contre des antigènes exposés en surface des cellules ovales et des canaux biliaires ou des hépatocytes. L'analyse par immunotransfert de protéines de cellules épithéliales non-parenchymateuses de foie de rat en culture révèle que l'anticorps anti-cytokératines d'hépatocytes de rat reconnaît une cytokératine de masse relative (Mr) 55 000, l'anticorps anti-cytokératines de sabots de vache réagit avec une cytokératine de Mr 52 000 et que les anticorps anti-vimentine réagissent avec la vimentine. Des analyses similaires avec les protéines de fibroblastes de rat en culture confirment que les anticorps anti-vimentine reconnaissent la vimentine. La coloration de coupes de foie congelé par double marquage en immunofluorescence utilisant les anticorps anti-cytokératines et anti-vimentine rélève un patron de réactivité unique pour chacune des différentes structures cellulaires du foie. Les hépatocytes sont colorés seulement par l'anticorps anti-cytokératine de Mr 55 000 tandis que les cellules sinusoidales réagissent seulement avec l'anti-vimentine. D'autre part, les cellules épithéliales des canaux biliaires et de la capsule de Glisson réagissent avec tous les anticorps anti-cytokératines et anti-vimentine. Cependant, la réaction de l'anticorps anti-vimentine avec les cellules des canaux biliaires est faible. Des analyses similaires sur des coupes de tissus ont montré que l'anticorps anti-cellules ovales réagit avec les structures des canaux biliaires mais non avec la capsule de Glisson tandis que l'anticorps anti-hépatocytes ne réagit qu'avec les cellules parenchymateuses. Cette même réactivité différentielle des anticorps anti-cytokératines et anti-vimentine se retrouve dans les cultures primaires d'hépatocytes, de cellules sinusoïdales et des canaux biliaires. Les anticorps utilisés ici permettent donc une distinction claire entre les cellules épithéliales non-parenchymateuses, les hépatocytes et les cellules sinusoïdales en culture. La microscopie par immunofluorescence indirecte sur des hépatocytes et des cellules des canaux biliaires non fixées ou fixées à la formaldehyde a confirmé que les anticorps anti-hépatocytes et anti-cellules ovales reconnaissent des éléments exposées en surface des types cellulaires respectifs. L'extension de cette méthode de typage à trois lignées établies de cellules hépatiques de rat ayant une morphologie épithéliale (RLEC, MLC et T51B) a permis de montrer que toutes ces lignées sont effectivement d'origine épithéliale puisqu'elles réagissent avec les anticorps anti-cytokératines. De plus, les réactions de ces lignées cellulaires avec les anticorps anti-vimentine donnent des patrons de réactivité équivalents à ceux des cellules épithéliales des structures biliaires et de la capsule de Glisson. De plus, les analyses avec les anticorps contre les composantes exposées en surface ont révélé une réaction de l'anticorps anti-cellules ovales sur les cellules T51B mais non sur les autres lignées cellulaires. Cela suggère fortement que les cellules T51B originent des cellules des canaux biliaires tandis que les autres lignées présentant le phénotype de cellules mésothéliales en culture origineraient de la capsule de Glisson.Cell of origin of distinct cultured rat liver epithelial cells, as typed by cytokeratin and surface component selective expression. Biochem. Cell Biol. 64, 788-802 The cell of origin of the nonparenchymal epithelioid cells that emerge in liver cell cultures is unknown. Cultures of rat hepatocytes and several types of nonparenchymal cells obtained by selective tissue dispersion procedures were typed with monoclonal antibodies to rat liver cytokeratin and vimentin, polyvalent antibodies to cow hoof cytokeratins and porcine lens vimentin, and monoclonal antibodies to surface membrane components of ductular oval cells and hepatocytes. Immunoblot analysis revealed that, in cultured rat liver nonparenchymal epithelial cells, the anti-rat hepatocyte cytokeratin antibody recognized a cytokeratin of relative mass (M,) 55 000 and the anti-cow hoof cytokeratin antibody reacted with a cytokeratin of M, 52 000, while the anti-vimentin antibodies detected vimentin in both cultured rat fibroblasts and nonparenchymal epithelial cells. Analyses on the specificity of anti-cytokeratin and anti-vimentin antibodies toward the various cellular structures of liver by double immunofluorescence staining of frozen tissue sections revealed unique reactivity patterns. For example, hepatocytes were only stained with anti-Mr 55 000 cytokeratin antibody, while the sinusoidal cells reacted only with the anti-vimentin antibodies. In contrast, epithelial cells of the bile ductular structures and mesothelial cells of the Glisson capsula reacted with all the anti-cytokeratin and anti-vimentin antibodies. It should be stressed, however, that the reaction of the anti-vimentin antibodies on bile ductular cells was weak. The same analysis on tissue sections using the anti-ductular oval cell antibody revealed that it reacted with bile duct structures but not with the Glisson capsula. The anti-hepatocyte antibody reacted only with the parenchymal cells. The differential reactivity of the anti-cytokeratin and anti-vimentin antibodies with the various liver cell compartments was confirmed in primary cultures of hepatocytes, sinusoidal cells, and bile ductular cells, indicating that the present panel of antibodies to intermediate filament constituants allowed a clear-cut distinction between cultured nonparenchymal epithelial cells, hepatocytes, and sinusoidal cells. Indirect immunofluorescence microscopy on nonfixed and paraformaldehyde-fixed cultured hepatocytes and bile ductular cells further confirmed that both anti-hepatocyte and anti-ductular oval cell antibodies recognized surface-exposed components on the respective cell types. By extending the use of this cell typing assay to three established rat liver cell lines of epithelioid morphology (RLEC, MLC, and T51B), we found that all of them reacted with the anti-cytokeratin antibodies, showing that they were indeed of epithelial cell origin. Moreover, these cells were stained with the anti-vimentin antibodies, which yielded differential reactivity patterns equivalent to those of epithelial cells from either the bile ductular structure or the Glisson capsula. Interestingly, the analysis with the antibodies against the exposed surface components revealed a reaction of the anti-ductular oval cell antibody on T51B cells, but not on the other cell lines. This strongly suggests that the T51B epithelial cell type is of bile ductular origin, while the latter cell lines, which exhibit a mesothelid-like phenotype in culture, most likely originate from the Glisson capsula

Identification, characterization and potent antitumor activity of ECO-4601, a novel peripheral benzodiazepine receptor ligand.

International audiencePURPOSE: ECO-4601 is a structurally novel farnesylated dibenzodiazepinone discovered through DECIPHER technology, Thallion's proprietary drug discovery platform. The compound was shown to have a broad cytotoxic activity in the low micromolar range when tested in the NCI 60 cell line panel. In the work presented here, ECO-4601 was further evaluated against brain tumor cell lines. Preliminary mechanistic studies as well as in vivo antitumor evaluation were performed. METHODS: Since ECO-4601 has a benzodiazepinone moiety, we first investigated if it binds the central and/or peripheral benzodiazepine receptors. ECO-4601 was tested in radioligand binding assays on benzodiazepine receptors obtained from rat hearts. The ability of ECO-4601 to inhibit the growth of CNS cancers was evaluated on a panel of mouse, rat and human glioma cell lines using a standard MTT assay. Antitumor efficacy studies were performed on gliomas (rat and human), human breast and human prostate mouse tumor xenografts. Antitumor activity and pharmacokinetic analysis of ECO-4601 was evaluated following intravenous (i.v.), subcutaneous (s.c.), and intraperitoneal (i.p.) bolus administrations. RESULTS: ECO-4601 was shown to bind the peripheral but not the central benzodiazepine receptor and inhibited the growth of CNS tumor cell lines. Bolus s.c. and i.p. administration gave rise to low but sustained drug exposure, and resulted in moderate to significant antitumor activity at doses that were well tolerated. In a rat glioma (C6) xenograft model, ECO-4601 produced up to 70% tumor growth inhibition (TGI) while in a human glioma (U-87MG) xenograft, TGI was 34%. Antitumor activity was highly significant in both human hormone-independent breast (MDA-MB-231) and prostate (PC-3) xenografts, resulting in TGI of 72 and 100%, respectively. On the other hand, i.v. dosing was followed by rapid elimination of the drug and was ineffective. CONCLUSIONS: Antitumor efficacy of ECO-4601 appears to be associated with the exposure parameter AUC and/or sustained drug levels rather than C (max). These in vivo data constitute a rationale for clinical studies testing prolonged continuous administration of ECO-4601

TLN-05220, TLN-05223, new Echinosporamicin-type antibiotics, and proposed revision of the structure of bravomicins

The deposited strain of the hazimicin producer, Micromonospora echinospora ssp. challisensis NRRL 12255 has considerable biosynthetic capabilities as revealed by genome scanning. Among these is a locus containing both type I and type II PKS genes. The presumed products of this locus, TLN-05220 (1) and TLN-05223 (2), bear a core backbone composed of six fused rings starting with a 2-pyridone moiety. The structures were confirmed by conventional spectral analyses including MS, and 1D and 2D NMR experiments. Comparison of both the 1H and 13C NMR data of the newly isolated compound with those of echinosporamicin and bravomicin A led us to propose a revision of the structure of the latter to include a 2-pyridone instead of the pyran originally postulated. Both compounds (1 and 2) possessed strong antibacterial activity against a series of gram-positive pathogens including several strains of methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci (VRE), and cytotoxic activities against several human tumor cell lines. The TLN compounds are the first of this group with reported anticancer activity.Peer reviewed: YesNRC publication: Ye

Synergistic activity of troxacitabine (Troxatyl™) and gemcitabine in pancreatic cancer-0

<p><b>Copyright information:</b></p><p>Taken from "Synergistic activity of troxacitabine (Troxatyl™) and gemcitabine in pancreatic cancer"</p><p>http://www.biomedcentral.com/1471-2407/7/121</p><p>BMC Cancer 2007;7():121-121.</p><p>Published online 3 Jul 2007</p><p>PMCID:PMC1948004.</p><p></p>xacitabine or gemcitabine either alone or in combination at a ratio of 1:100 of gemcitabine vs. troxacitabine, for 72 h, after which cells were harvested by trypsinization and their numbers determined using electronic particle counting. Each data point represents the mean ± SD of three determinations. Gemcitabine (open squares), troxacitabine (open inverted triangle), gemcitabine + troxacitabine (open circle). The GIvalues for exposures to troxacitabine and gemcitabine alone are given in Table 2

Synergistic activity of troxacitabine (Troxatyl™) and gemcitabine in pancreatic cancer-4

<p><b>Copyright information:</b></p><p>Taken from "Synergistic activity of troxacitabine (Troxatyl™) and gemcitabine in pancreatic cancer"</p><p>http://www.biomedcentral.com/1471-2407/7/121</p><p>BMC Cancer 2007;7():121-121.</p><p>Published online 3 Jul 2007</p><p>PMCID:PMC1948004.</p><p></p>aCa-2, AsPc-1 and Capan-2 cells. The individual doses of gemcitabine and troxacitabine to achieve 90% (straight line) growth inhibition (Fa = 0.90), 75% (hyphenated line) growth inhibition (Fa = 0.75), and 50% (dotted line) growth inhibition (Fa = 0.50) were plotted on the x- and y-axes. Combination index (CI) values calculated using Calcusyn software is represented by points above (indicate antagonism between drugs) or below the lines (indicate synergy). (X symbol) ED(plus sign) EDand (open dotted circle ) ED