Alterations in specific signal transduction pathways may explain the increased expression of proinflammatory cytokines seen in inflammatory diseases such as psoriasis. We reveal increased TNF-alpha protein expression, but similar TNF-alpha mRNA levels, in lesional compared with nonlesional psoriatic skin, demonstrating for the first time that TNF-alpha expression in lesional psoriatic skin is regulated posttranscriptionally. Increased levels of activated MAPK-activated protein kinase 2 (MK2) together with increased MK2 kinase activity were found in lesional compared with nonlesional psoriatic skin. Immunohistochemical analysis showed that activated MK2 was located in the basal layers of the psoriatic epidermis, whereas no positive staining was seen in nonlesional psoriatic skin. In vitro experiments demonstrated that both anisomycin and IL-1beta caused a significant activation of p38 MAPK and MK2 in cultured normal human keratinocytes. In addition, TNF-alpha protein levels were significantly up-regulated in keratinocytes stimulated with anisomycin or IL-1beta. This increase in TNF-alpha protein expression was completely blocked by the p38 inhibitor, SB202190. Transfection of cultured keratinocytes with MK2-specific small interfering RNA led to a significant decrease in MK2 expression and a subsequent significant reduction in the protein expression of the proinflammatory cytokines TNF-alpha, IL-6, and IL-8, whereas no change in the expression of the anti-inflammatory cytokine IL-10 was seen. This is the first time that MK2 expression and activity have been investigated in an inflammatory disease such as psoriasis. The results strongly suggest that increased activation of MK2 is responsible for the elevated and posttranscriptionally regulated TNF-alpha protein expression in psoriatic skin, making MK2 a potential target in the treatment of psoriasis.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D3, inhibits the proliferation of prostate cancer cells. However, the molecular mechanisms by which 1,25(OH)2D3 inhibits the proliferation of these cells remain to be fully elucidated. In this study, we used microarray technology to identify target genes of 1,25(OH)2D3 in androgen-responsive prostate cancer LNCaP cells. 1,25(OH)2D3 up-regulated CCAAT/enhancer-binding protein delta (C/EBPdelta) by approximately 5-fold in these cells. Knockdown of C/EBPdelta expression by RNA interference showed that C/EBPdelta is essential for the significant growth inhibition of LNCaP cells in response to 1,25(OH)2D3 treatment. Moreover, we found that 1,25(OH)2D3 induced C/EBPdelta in other cancer cells, including the estrogen receptor (ER)-expressing MCF-7 and T47D breast cancer cells that are sensitive to the growth inhibitory effects of 1,25(OH)2D3. On the other hand, 1,25(OH)2D3 was not able to induce C/EBPdelta in either androgen receptor-negative PC-3 and DU145 or ER-negative breast cancer MDA-MB-231 cells that were relatively resistant to growth inhibition by 1,25(OH)2D3. Furthermore, forced expression of C/EBPdelta in prostate cancer LNCaP as well as breast cancer MCF-7 and T47D cells dramatically reduced their clonal growth. Taken together, forced expression of C/EBPdelta in cancer cells may be a promising therapeutic strategy.

To overcome pharmacokinetic and solubility problems observed in early clinical trials with the potent anticancer compound CHS828, we synthesised a series of prodrugs with improved properties. The best compound obtained was EB1627, with a tetraethyleneglycol moiety attached to the parent drug via a carbonate linkage. This compound was found soluble enough to be given i.v. and the drug was rapidly released in vivo exerting a very potent inhibitory activity alone and in combination with known cytostatics (etoposide) in animal models in vivo.

Experimental and clinical studies indicate that low molecular weight heparins (LMWH) may inhibit cancer and/or metastasis. The purpose of this study was to investigate whether it is possible to design non-anti-coagulant, anti-metastatic compounds based on heparin. The LMWH Tinzaparin and a series of non-anti-coagulant (NAC) heparin derivatives, varying in size from 2,500 to 10,000 Da, were tested for their anti-metastatic activity in an experimental B16F10 metastasis model. The most promising NAC heparin drug candidate and Tinzaparin were further evaluated in B16F10 model with spontaneous metastasis from a primary subcutaneous tumor. In the experimental model, Tinzaparin, NAC2500, and NAC6000 were inactive whereas both NAC8000 and NAC10000 significantly inhibited the number of induced experimental metastases by 69 and 73%, respectively. NAC8000 was chosen over NAC10000 for further studies because of its lower molecular weight with an expected better bioavailability. In the spontaneous model, Tinzaparin had no inhibitory effect on metastatic activity. In contrast, NAC8000 significantly inhibited the number of metastases by 58%. Neither Tinzaparin nor NAC8000 inhibited primary subcutaneous tumor growth. Together, these results indicate that the anti-metastatic effect of heparin derivatives is not a result of anti-coagulant activity. The non-anti-coagulant NAC8000 specifically inhibits early establishment of tumor cells, but not primary tumor growth. Therefore, NAC8000 is a promising non-anti -coagulant compound for preventing tumor metastasis.

Introduction The cytochrome P450 mitochondrial enzyme 25-hydroxyvitamin D-3 1 alpha-hydroxylase (1 alpha-hydroxylase) of renal tubule cells hydroxylates the major circulating form of vitamin D (25(OH) D-3) to the active systemic hormone 1,25(OH)(2)D-3. Local production of 1,25( OH)(2)D-3 appears to occur also at other sites where 1 alpha-hydroxylase is expressed for autocrine/ paracrine regulation. To reduce risks of hypercalcemia during treatment with vitamin D, we have previously suggested use of non-1 alpha-hydroxylated vitamin D analogues to target tissues where 1 alpha-hydroxylase is expressed, including the parathyroid glands in secondary hyperparathyroidism. The present study was undertaken to examine expression of 1 alpha-hydroxylase in breast cancer and to investigate whether a non-1 alpha-hydroxylated vitamin D analogue displayed biological function. In addition, expression of the 25-hydroxyvitamin D-3 24-hydroxylase (24-hydroxylase) and the vitamin D receptor (VDR) was investigated. Methods The expression of 1 alpha-hydroxylase, 24-hydroxylase and VDR was investigated in breast cancer specimens ( n = 19) and normal breast tissues ( n = 10) by immunohistochemistry and/or RT-PCR. Consecutive cryosections of 6 mu m essentially free of immune cells were used in the analyses. The effect of vitamin D analogues on transcriptional activation was analyzed in transiently transfected MCF-7 breast cancer cells. Results 1 alpha-hydroxylase protein was demonstrated in 79% and 100% of breast cancer specimens and normal breast, respectively. The overall relative mRNA levels of 1 alpha-hydroxylase and 24-hydroxylase in normal breast compared to breast tumors were: 1 alpha-hydroxylase, 1 +/- 0.07 versus 0.7 +/- 0.05, respectively ( p < 0.001); 24-hydroxylase, 1 +/- 0.08 verus 2.1 +/- 0.2, respectively ( p < 0.001). The VDR was expressed in 95% of the tumors as expected, with mRNA levels of 1 +/- 0.09 and 1.4 +/- 0.12 ( p < 0.05) in breast cancer and normal breast, respectively. The ketoconazole- sensitive transcription activation potential of the non-1 alpha-hydroxylated vitamin D analogue prodrug of EB1089 (EB1285) was demonstrated in MCF-7 cells, which express 1 alpha-hydroxylase. The activity of EB1285 was about 20% of 1,25(OH)(2)D-3. Conclusion These results demonstrate nearly normal expression levels of 1 alpha-hydroxylase, 24-hydroxylase and VDR in the majority of investigated breast cancer specimens. A non-1 alpha-hydroxylated vitamin D analogue displayed activity in breast cancer cells. Such analogues may present future therapeutic options for proliferative disorders where 1 alpha-hydroxylase is expressed.

The cytochrome P450 mitochondrial enzyme 25-hydroxyvitamin D3 1α-hydroxylase (1α-hydroxylase) of renal tubule cells hydroxylates the major circulating form of vitamin D (25(OH)D3) to the active systemic hormone 1,25(OH)2D3. Local production of 1,25(OH)2D3 appears to occur also at other sites where 1α-hydroxylase is expressed for autocrine/paracrine regulation. To reduce risks of hypercalcemia during treatment with vitamin D, we have previously suggested use of non-1α-hydroxylated vitamin D analogues to target tissues where 1α-hydroxylase is expressed, including the parathyroid glands in secondary hyperparathyroidism. The present study was undertaken to examine expression of 1α-hydroxylase in breast cancer and to investigate whether a non-1α-hydroxylated vitamin D analogue displayed biological function. In addition, expression of the 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase) and the vitamin D receptor (VDR) was investigated.

Seocalcitol (EB 1089) is under development for the treatment of hepato-cellular carcinoma (HCC). The tissue distribution of 3H-seocalcitol was investigated in comparison to 3H-calcitriol in rats. Quantitative whole-body autoradiography was used to quantify the tissue distribution. The greatest difference in distribution between the two compounds was observed in the bloodstream. For most tissues the ratio seocalcitol/calcitriol varied between 0.2 and 3.1. The concentration of radioactivity in the liver was almost the same for the two compounds. For seocalcitol the concentration in the liver was 10 times higher than in serum. Assuming that the liver/serum concentration ratio is the same in rats and humans, the concentration of seocalcitol in the human liver is expected to be higher than the concentration resulting in more than 50% inhibition of cancer cell proliferation, and thus pharmacologically effective in HCC. It is questionable whether calcitriol would be present in the human liver in sufficient concentrations to be effective for the treatment of HCC, as the antiproliferative activity of calcitriol is generally more than 10-fold lower compared to that of seocalcitol and as calcitriol can only be administered at a dose that is ca. three-fold lower than the dose of seocalcitol.

CHS 828 is a pyridyl cyanoguanidine with promising antitumor activity both in vitro and in vivo, and has previously been found especially active against tumor cells obtained from patients with B cell chronic lymphocytic leukemia. In the present study the cytotoxic effect in vitro of CHS 828 was investigated on a panel of 10 human myeloma cell lines using the fluorometric microculture cytotoxicity assay. CHS 828 induced a concentration-dependent, but variable decrease in tumor cell survival in the cell line panel with inhibitory concentrations 50% (IC50) in the range 0.01-0.3 microM. These concentrations are below those achievable in vivo. There was no detectable dependence on P-glycoprotein-mediated or GSH-associated drug resistance and the drug showed low to moderate cross-resistance with standard drugs, including melphalan, vincristine and doxorubicin. Furthermore, sensitivity to CHS 828 showed no apparent relationship to growth factor dependence, tumor progression or phenotypic variability. CHS 828 was also tested in vivo using a hollow fiber model in rats with three of the cell lines. The results indicate a high cytotoxic activity of CHS 828. Overall, the results show a high cytotoxic activity of CHS 828 in the myeloma models, which might warrant its further development against myeloma.

We recently described an in vivo angiogenesis assay for rats--an optimized Matrigel plug assay based on subcutaneously implanted chambers with fixed volume and shape. Here we examine the possibility of switching the host animal from rat to mouse, thereby reducing the requirement for test compound an order of magnitude. The chambers consist of a plexiglas ring with a 0.2 ml volume and two nylon net filters. Chambers containing growth factor-reduced Matrigel supplemented with basic fibroblast growth factor (bFGF) were subcutaneously implanted in the right flank of three different strains of mice; BALB/c, C57BL/6J, and NMRI-nu. On day 10 post-implantation: i) each chamber was taken out, ii) a picture of the induced angiogenic response was taken, and iii) the redness of the chamber content was quantified by computer image analysis. The level of bFGF-induced angiogenesis in the mouse assay was lower than in the previously published rat assay. Importantly, the background angiogenesis in mice in chambers containing Matrigel alone was correspondingly decreased. Therefore, a more sensitive threshold for the computer image analysis was used. In all three strains of mice, bFGF-induced angiogenesis was significantly increased compared to Matrigel alone. Furthermore, the positive anti-angiogenic control compound TNP-470 (10 mg/kg/d s.c.) completely inhibited the bFGF-induced angiogenesis. The in vivo chamber angiogenesis assay allows quantitative analysis of angiogenic and anti-angiogenic activity in mice. The model is very robust and only little influenced by the choice of mouse strain.

Several vitamin D analogues, with reduced hypercalcemic and hyperphosphatemic toxicity at therapeutic dosages, are in clinical use for prevention and treatment of secondary hyperparathyroidism (HPT) in chronic renal failure. We have performed a first in vitro evaluation of five vitamin D analogues displaying less calcemic activity in normal rats, considerably more antiproliferative ability and higher transcription activation potential than 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), with the future prospects in mind to identify even more effective and less calcemic vitamin D analogues for treatment of HPT. The vitamin D analogues EB1089 and GS1590 have polyunsaturated side-chains, whereas HEP187, MC1598 and CB1393 display altered stereochemistry at carbon 20. In primary cultures of bovine parathyroid cells EB1089, GS1590, CB1393 and MC1598 as well as the comparative controls 1,25(OH)(2)D(3), 22-oxacalcitriol (OCT, maxacalcitol), 19-nor-1,25(OH)(2)D(2) (paricalcitol) and 1alpha(OH)D(2) (doxercalciferol) significantly suppressed PTH secretion or reduced PTH mRNA level at 10(-8), 10(-10), and 10(-11)M for all compounds except for MC1598 at the lowest concentration. The analogue HEP187 displayed no PTH suppressive activity. We conclude that the vitamin D analogues EB1089, GS1590 and CB1393 may be suitable for treatment of hyperparathyroidism secondary to uremia and that further evaluation in vivo should be considered.

Although both the antiapoptotic function of survivin and vitamin D 3 (VD3)-mediated cell growth inhibition and apoptosis have been extensively studied, it is not known whether survivin plays a role in VD3 compound-mediated cell growth inhibition and apoptosis induction. Using an isogenic model of MCF-7 breast adenocarcinoma cells (MCF-7E and MCF-7L sublines that are sensitive and resistant to VD3 compounds), we found that VD3 compounds effectively downregulated survivin in VD3-sensitive MCF-7E cells, which was associated with VD3-induced apoptosis. In contrast, VD3 compounds failed to downregulate survivin in VD3-resistant MCF-7L cells, which showed resistant to VD3-induced apoptosis. However, inhibition of survivin expression by small interfering RNA (siRNA) induced cell death per se and further sensitized VD3-induced apoptosis in MCF-7L cells, indicating that the inability of these cells to respond to VD3 is due to the failure to downregulate survivin. Forced expression of survivin not only blocked VD3-mediated G1 cell accumulation but also increased S and G2/M cell populations. VD3 treatment rapidly triggered the activation of p38 MAPK signaling in MCF-7E cells but not in MCF-7L cells. Moreover, inhibition of p38 activation diminished VD3-mediated survivin inhibition and partially rescued VD3-induced cell death. We further showed that VD3 increased the expression of TGF β 1 and TGF β receptor 2, and that blocking the function of TGF β receptor 2 diminished VD3 compound-mediated survivin downregulation. Thus, we propose that the VD3 compound-induced growth inhibition and apoptosis induction are at least partially dependent on survivin downregulation via VD3-induced TGF β signaling and the activation of p38 MAPK pathway. Targeting survivin through these pathways may lead to novel applications for cancer therapeutics.

The cyanoguanidine CHS 828 has shown promising antitumor properties and is currently in early clinical trials, although the mechanism of action still is largely unknown. In this study, resistant sublines of the histiocytic lymphoma cell line U-937 GTB and the myeloma line RPMI 8226 were developed by culturing under gradually increasing concentrations of CHS 828 until reaching 25 times the parental line EC50s. The new phenotypes demonstrate more than 400-fold resistance to CHS 828 and cross-resistance to six cyanoguanidine analogs, but no resistance to nine standard drugs of different mechanistic classes or to the cytotoxic guanidines m-iodobenzylguanidine and methylglyoxal-bis(guanylhydrazone). The resistant phenotypes were stable for several months even if cultivated in drug-free medium and no difference in proliferation, ultrastructural or morphologic appearance in the sublines could be detected. Neither was decreased accumulation of tritium-labeled CHS 828 observed. Furthermore, the new U-937 phenotype was not accompanied by changes in differentiation or an altered cell-cycle distribution. In the myeloma cell line, esterase activity was shown to be moderately enhanced. Two-dimensional protein electrophoresis was undertaken to unmask possible resistance-mediating proteins and/or the target molecule(s) for CHS 828. In the myeloma cell line, λ light chain immunoglobulin (down-regulated) and a fatty acid-binding protein (up-regulated) were identified. The findings presented here indicate that development of specific cellular alterations is responsible for the gained CHS 828 resistance.

Seocalcitol (EB 1089) is underdevelopment for the treatment of hepato-cellular carcinoma (HCC). The tissue distribution of H-3-seocalcitol was investigated in comparison to H-3-calcitriol in rats. Quantitative whole-body autoradiography was used to quantify the tissue distribution. The greatest difference in distribution between the two compounds was observed in the bloodstream. For most tissues the ratio seocalcitol/calcitriol varied between 0.2 and 3.1. The concentration of radioactivity in the liver was almost the same for the two compounds. For seocalcitol the concentration in the liver was 10 times higher than in serum. Assuming that the liver/serum concentration ratio is the same in rats and humans, the concentration of seocalcitol in the human liver is expected to be higher than the concentration resulting in more than 50% inhibition of cancer cell proliferation, and thus pharmacologically effective in HCC. It is questionable whether calcitriol would be present in the human liver in sufficient concentrations to be effective for the treatment of HCC, as the anti proliferative activity of calcitriol is generally more than 10-fold lower compared to that of seocalcitol and as calcitriol can only be administered at a dose that is ca. three-fold lower than the dose of seocalcitol. (C) 2004 Elsevier Ltd. All rights reserved.

We wish to report the synthesis and structure-activity relationship (SAR) of a series of 4-aminobenzophenones, as a novel compound class with high antiinflammatory activity. Our initial lead, (4-[(2-aminophenyl)amino]phenyl)(phenyl)methanone (3), was systematically optimized and resulted in compounds that potently inhibited the release of the proinflammatory cytokines IL-1beta and TNF-alpha in human peripheral blood mononuclear cells stimulated by LPS. One of the most potent compounds, among others, was (4-[(2-aminophenyl)amino]-2-chlorophenyl)(2-methylphenyl)methanone (45) with IC(50) values of 14 and 6 nM for the inhibition of IL-1beta and TNF-alpha, respectively. Furthermore, we found these types of compounds to be potent and selective p38 MAP kinase inhibitors, e.g. 45 had an IC(50) value of 10 nM. Molecular modeling was used to rationalize our SAR data and to propose a model for the interaction of compound 45 with the p38 MAP kinase. The model involved a favorable hydrogen bond between the carbonyl group of the benzophenone and the NH of Met-109, positioning ring A in the hydrophobic pocket I of the enzyme. Good antiinflammatory effects were demonstrated in two murine models of dermatitis after topical application (oxazolone and TPA model).

BACKGROUND:Studies have identified analogs of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], which in vitro are 10- to 3,000-fold more active than 1,25(OH)2D3. We compared in vivo the anti-cancer activity of three potent vitamin D3 analogs and 1,25(OH)2D3 at near to each of their maximal tolerated dose (MTD).MATERIALS AND METHODS:Human LNCaP prostate cancer xenografts were grown in nude mice and the animals were treated with intraperitoneal injections of either diluant; 1,25(OH)2D3; 1,25-Dihydroxy-20epi-22-oxa-24,26,27-trisho-mocholecalciferol (KH 1060); 1,25-Dihydroxy-22E,24E-diene-24,26,27-trishomocholecalciferol (EB 1039); and 1,25-Dihydroxy-16-ene-24-oxo-19-norcholecalciferol (RO 26-9114). Tumor sizes were measured weekly and tumor weights were measured at autopsy on the 12th week.RESULTS:Each of the analogs equally and markedly inhibited growth of the prostate cancer xenografts. The 1,25(OH)2D3 initially inhibited growth but, by the time of sacrifice, the tumors were nearly the same size as diluant controls. The histological examination of the tumors showed that the analogs produced tumor necrosis and microcalcification. None of the mice developed hypercalcemia, which is the major toxicity of vitamin D3 compounds.CONCLUSION:The MTD of the analogs varied by 400-fold but each had similar efficacy suggesting that, when choosing a vitamin D analog for clinical study, overall efficacy without toxicity is more important than the total amount of the compound that can be administered. In summary, we have identified three vitamin D analogs that show marked potency in vivo to inhibit growth of human prostate cancer xenografts; each had no detectable toxicity. This study should help lay the foundation for clinical studies.

Efficient in vitro and in vivo angiogenesis assays, to assess and compare anti-angiogenic activity are a prerequisite for the discovery and characterization of anti-angiogenic targets. Here we describe an optimized Matrigel plug assay based on subcutaneously implanted chambers and two fast and reproducible measuring techniques. Plexiglas ring/nylon net filter-chambers (0.2 ml) containing growth factor-reduced Matrigel and 300 ng basic fibroblast growth factor (bFGF) were subcutaneously implanted into the right flank of rats. Chamber angiogenesis was scored on day 5 and day 10 post-implantation by computer image analysis of the chamber, and by optical density reading at 415 nm of a PBS solution of the chamber content. bFGF significantly induced chamber angiogenesis and histological examination confirmed that numerous blood vessels were present in the bFGF-induced chambers. The anti-angiogenic control compound TNP-470 (10 mg/kg/d s.c.) completely inhibited the bFGF-induced angiogenesis. In contrast, the anti-inflammatory or immuno-suppressive compounds cyclosporin A (15 mg/kg/d p.o.), indomethacin (1 mg/kg/d p.o.), and prednisolone (5 mg/kg/d p.o.) showed no anti-angiogenic activity, indicating that the bFGF-induced angiogenesis was not driven by an inflammatory response or by a foreign body reaction. Finally, two candidate anti-angiogenic compounds were tested in the assay. Continuous low-dose therapy with cyclophosphamide (25 mg/kg/d p.o.) significantly inhibited bFGF-induced angiogenesis, whereas 1alpha,25-dihydroxyvitamin D3 (0.5 micro g/kg/d p.o.) showed no significant anti-angiogenic activity. In conclusion, this in vivo chamber angiogenesis assay is a useful new tool for drug evaluation as well as research into anti-angiogenesis.