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Abstract Reliable quantitative evaluation of molecular pathways is critical for both drug discovery and treatment monitoring. We have modified the CAM assay to quantitatively measure vascular density, endothelial proliferation, and changes in protein expression in response to anti-angiogenic and pro-angiogenic agents. This improved CAM assay can correlate changes in vascular density with changes seen on a molecular level. We expect that these described modifications will result in a single in vivo assay system, which will improve the ability to investigate molecular mechanisms underlying the angiogenic response.

Reliable quantitative evaluation of molecular pathways is critical for both drug discovery and treatment monitoring. We have modified the CAM assay to quantitatively measure vascular density, endothelial proliferation, and changes in protein expression in response to anti-angiogenic and pro-angiogenic agents. This improved CAM assay can correlate changes in vascular density with changes seen on a molecular level. We expect that these described modifications will result in a single in vivo assay system, which will improve the ability to investigate molecular mechanisms underlying the angiogenic response. [ABSTRACT FROM AUTHOR]

We have found that the synthetic compound CC-5079 potently inhibits cancer cell growth in vitro and in vivo by a novel combination of molecular mechanisms. CC-5079 inhibits proliferation of cancer cell lines from various organs and tissues at nanomolar concentrations. Its IC(50) value ranges from 4.1 to 50 nmol/L. The effect of CC-5079 on cell growth is associated with cell cycle arrest in G(2)-M phase, increased phosphorylation of G(2)-M checkpoint proteins, and apoptosis. CC-5079 prevents polymerization of purified tubulin in a concentration-dependent manner in vitro and depolymerizes microtubules in cultured cancer cells. In competitive binding assays, CC-5079 competes with [(3)H]colchicine for binding to tubulin; however, it does not compete with [(3)H]paclitaxel (Taxol) or [(3)H]vinblastine. Our data indicate that CC-5079 inhibits cancer cell growth with a mechanism of action similar to that of other tubulin inhibitors. However, CC-5079 remains active against multidrug-resistant cancer cells unlike other tubulin-interacting drugs, such as Taxol and colchicine. Interestingly, CC-5079 also inhibits tumor necrosis factor-alpha (TNF-alpha) secretion from lipopolysaccharide-stimulated human peripheral blood mononuclear cells (IC(50), 270 nmol/L). This inhibitory effect on TNF-alpha production is related to its inhibition of phosphodiesterase type 4 enzymatic activity. Moreover, in a mouse xenograft model using HCT-116 human colorectal tumor cells, CC-5079 significantly inhibits tumor growth in vivo. In conclusion, our data indicate that CC-5079 represents a new chemotype with novel mechanisms of action and that it has the potential to be developed for neoplastic and inflammatory disease therapy.

Immunomodulatory drugs (IMiDs) are orally available small molecules that potently inhibit tumor necrosis factor-alpha (TNF-alpha) production by lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (HuPBMCs) but enhance secretion of such cytokines as interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) by stimulated T cells. The mechanism of cytokine regulation by IMiDs has not yet been determined. In the present study, we investigated the effects of one of the IMiDs, CC-4047 (Actimid, Celgene, Warren, NJ), on synthesis of IL-2 protein and mRNA and on the activity and expression of transcription factors. Treatment with CC-4047 enhances the secretion of IL-2 protein and the expression of IL-2 mRNA in a dose-dependent and time-dependent manner. In T cells stimulated with phorbol myristate acetate (PMA)/ionomycin, CC-4047 enhanced the DNA-binding activity of activated protein-1 (AP-1) but not NF-kappaB, Octomer-1 (OCT-1), or NFAT by 2-fold and 4-fold after an incubation time of 1 and 3 h, respectively. Luciferase reporter assays in Jurkat cells showed similar effects on transcription factor activity. Using in vitro kinase activity assays, we also showed that CC-4047 enhances the activity of protein kinase C-theta (PKC-theta) in stimulated T cells. The secreted IL-2 from HuPBMCs was shown to activate natural killer (NK) cells to lyse their target cell line K562. Taken together, our results demonstrate that the IMiDs exert their effects at least in part by activating PKC-theta and acting on AP-1 DNA-binding activity in T cells, resulting in augmented IL-2 synthesis and activation of IL- 2-dependent downstream effectors, such as NK cells.

Immunomodulatory drugs (IMiDs) are potent inhibitors of TNF-alpha and IL-1beta and elevators of IL-10 production in LPS-stimulated human PBMC. They are currently in clinical trials for various diseases, including multiple myeloma, myelodysplastic syndrome, and melanoma. In the present study, we have investigated the effects of thalidomide, CC-5013 and CC-4047 on the expression of COX-2 by stimulated PBMC. Our results show that thalidomide and IMiDs inhibited the expression of COX-2 but not the COX-1 protein in LPS-TNF-alpha and IL-1beta stimulated PBMC and shortened the half-life of COX-2 mRNA in a dose-dependent manner. They also inhibited the synthesis of prostaglandin E2 from LPS-stimulated PBMC. While anti-TNF-alpha or IL-1beta neutralizing antibodies had no effect on COX-2 expression, anti-IL-10 neutralizing antibody elevated the expression of COX-2 mRNA, and protein from treated PBMC. These data suggest that the anti-inflammatory and anti-tumor effects of IMiDs may be due in part to elevation of IL-10 production and its subsequent inhibition of COX-2 expression.

Selective cytokine inhibitory drugs (SelCIDs) are a novel class of phosphodiesterase 4 inhibitors discovered during a thalidomide analog discovery program. These analogs were evaluated for their ability to inhibit tumor angiogenesis, vascularity, and growth. Two analogs (CC-7034 and CC-9088) were identified that had enhanced antiangiogenic activity in Matrigel assays compared with parental thalidomide. These analogs also inhibited the growth of established K1735 and RENCA murine tumors. Tumors whose growth was suppressed by SelCID treatment exhibited decreased vessel density together with increased tumor cell hypoxia and death. The decrease in vascularity produced by SelCID treatment is attributed to a selective loss of vessels devoid of pericyte coverage, suggesting that these agents target immature tumor vessels. That tumor cell death was localized to relatively avascular or hypoxic areas, coupled with the fact that none of the analogs was cytotoxic in vitro against the tumor cells, demonstrates that these analogs are novel antivascular agents with potent antitumor activity.

Background: Activation of the NF-kappaB transcription factor has been previously demonstrated in two androgen receptor negative prostate cancer cell lines. We wished to extend this work to additional prostate cancer cells and to characterize the mechanisms responsible for constitutive NF-kappaB activation., Methods: Electrophoretic mobility shift assays were performed to measure NF-kappaB DNA-binding activity in prostate cancer cell lines, and immunohistochemistry was performed to detect nuclear localization of NF-kappaB in prostate cancer tissues. Western blot analysis was used to study the status of IkappaBalpha. Transient transfection assays were employed to characterize the contributions of IkappaB kinase (IKK), MAPK kinase kinases (MAPKKKs), androgen receptor (AR), and tyrosine phosphorylation to the constitutive activation of NF-kappaB in the prostate cancer cell lines., Results: Constitutive NF-kappaB activity was observed in AR-negative cell lines as well as in the prostate cancer patient samples, but was not present in AR positive cells. A "super-repressor" IkappaBalpha, as well as dominant negative forms of IKKbeta and NF-kappaB-inducing kinase (NIK), and tyrosine kinase inhibition were able to suppress NF-kappaB activity in the cells with constitutive activation., Conclusions: The constitutive activation of NF-kappaB observed in prostate cancer cells is likely due to a signal transduction pathway involving tyrosine kinases, NIK, and IKK activation., (Copyright 2002 Wiley-Liss, Inc.)