Retrovirus-transformed erythroleukemia cells induce central nervous system failure in a new syngeneic mouse model of meningeal leukemia.

Lack of suitable mouse models for central nervous system (CNS)-associated leukemias has hindered mechanism-guided development of therapeutics. By transplanting retrovirus-transformed mouse erythroleukemia cells into syngeneic mice, we developed a new animal model of meningeal leukemia associated with rapid paralysis. Necropsy revealed massive proliferation of the leukemic cells in the bone marrow (BM) followed by pathological angiogenesis and invasion of the leukemic cells into the meninges of the CNS. Further analysis demonstrated that the erythroleukemia cells secreted high levels of VEGF and preferentially adhered in vitro to fibronectin. This unique animal model for meningeal leukemia should facilitate studies of engraftment and proliferation of leukemic cells in the BM and their invasion of the CNS as well as pre-clinical evaluation of experimental therapeutics for CNS-associated leukemias.

Exploring the diversity of marine-derived fungal polyketide synthases.

Using an approach based on polymerase chain reaction (PCR), we examined the diversity of polyketide synthase (PKS) genes present in 160 marine fungal isolates, representing 142 species. We obtained ketosynthase (KS) domain PCR products from 99 fungal isolates, representing Dothideomycetes, Sordariomycetes, Eurotiomycetes, and incertae sedis. Sequence similarity searches and phylogenetic analysis of 29 marine partial-KS-encoding sequences revealed domains predicted to encode reducing, nonreducing, and 6-methylsalicylic acid PKSs. Bioinformatic analysis of an alignment of the KS sequences from marine-derived fungi revealed no unique motifs in this region. However, several specificity-determining positions were apparent between fungal 6-methylsalicylic acid PKSs as compared with either reducing or nonreducing PKSs. Evaluation of these positions in the context of a modelled three-dimensional protein structure highlighted their potential use as PKS classification markers. Evaluating primer-binding sites was necessary to obtain KS domain fragments from putative PKSs while maintaining a level of sequence information adequate to properly classify and characterize them.

Evaluation of human fetal bone implants in SCID mice as a model of prostate cancer bone metastasis.

The metastasis of prostate cancer cells to the bone marrow constitutes the major source of morbidity and mortality in prostate cancer. Studying this process has been hampered by the lack of preclinical models to evaluate novel therapeutics and to study the biology of the disease. One proposed model utilizes human fetal bone implants to serve as the target for prostate cancer cells injected via the tail vein. We employed this model to test the ability of zoledronic acid to prophylax and to treat bone metastases. To improve the rate of bone metastasis, we used two bone implants instead of one to evaluate the cell lines PC3 and PC3M, a more metastatic subline. For this purpose we generated the novel cell line PC3EGFPLuc, which can be used for luminescence and/or fluorescence imaging in vivo. We did not observe bone implant metastases in 52 mice, with 90 bone implants following tail vein injection of 1x10(6) PC3 or PC3M cells. Soft tissue lesions in the buttocks and hind limbs as well as cellular growth in the hindlimbs were observed via bioluminescence imaging. This evidence together with literature findings suggests that this model produces artifactual 'bone metastasis' lesions.

Small molecule-mediated anti-cancer therapy via hypoxia-inducible factor-1 blockade.

Despite reservations regarding potential toxicities, small molecule-mediated blockade of the hypoxia-inducible factor-1 transcription factor has emerged as a viable anti-cancer strategy in vivo. Recent experiments by Welsh et al. revealed unprecedented anti-tumor responses of various aggressive solid tumors to the HIF-1-inhibitory small molecule drug PX-478. Compared with other anti-cancer drugs, PX-478 had markedly improved regression, growth delay and log10 cell kill profiles, particularly against large tumors that are normally refractory to small molecule drug therapy. Importantly, pharmacokinetic and toxicity profiles were within acceptable limits, providing rationale for the clinical development of HIF-1 inhibitors in general. Though the mechanism of action for PX-478 is not completely understood, inhibition of glycolysis rather than angiogenesis appeared to be the primary mode of anti-cancer activity.

Antitumor effects of thalidomide analogs in human prostate cancer xenografts implanted in immunodeficient mice.

PURPOSE: Thalidomide has demonstrated clinical activity in various malignancies including androgen-independent prostate cancer. The development of novel thalidomide analogs with better activity/toxicity profiles is an ongoing research effort. Our laboratory previously reported the in vitro antiangiogenic activity of the N-substituted thalidomide analog CPS11 and the tetrafluorinated analogs CPS45 and CPS49. The current study evaluated the therapeutic potential of these analogs in the treatment of prostate cancer in vivo. EXPERIMENTAL DESIGN: Severely combined immunodeficient mice bearing s.c. human prostate cancer (PC3 or 22Rv1) xenografts were treated with the analogs at their maximum tolerated doses. Tumors were then excised and processed for ELISA and CD31 immunostaining to determine the levels of various angiogenic factors and microvessel density (MVD), respectively. RESULTS: CPS11, CPS45, and CPS49 induced prominent and modest growth inhibition in PC3 and 22Rv1 tumors, respectively. Thalidomide had no effect on tumor growth in either xenograft. Vascular endothelial growth factor and basic fibroblast growth factor levels were not significantly altered by any of the thalidomide analogs or thalidomide in both PC3 and 22Rv1 tumors. CPS45, CPS49, and thalidomide significantly reduced PC3 tumor platelet-derived growth factor (PDGF)-AA levels by 58-82% (P < 0.05). Interestingly, treatment with the analogs and thalidomide resulted in differential down-regulation (>/=1.5-fold) of genes encoding PDGF and PDGF receptor isoforms as determined by DNA microarray analysis. Intratumoral MVD of 22Rv1 xenografts was significantly decreased by CPS45 and CPS49. CPS49 also reduced MVD in PC3 xenografts. CONCLUSIONS: Thalidomide analogs CPS11 and 49 are promising anti-cancer agents. PDGF signaling pathway may be a potential target for these thalidomide analogs. Detailed microarray and functional analyses are under way with the aim of elucidating the molecular mechanism(s) of action of these thalidomide analogs.

Thalidomide.

Despite its history as a human teratogen, thalidomide is emerging as a treatment for cancer and inflammatory diseases. Although the evolution of its clinical application could not have been predicted from the tragedy associated with its misuse in the past, its history serves as a lesson in drug development that underscores the need to understand the molecular pharmacology of a compound's activity, including associated toxicities. Here, we summarise the applications for thalidomide with an emphasis on clinical trials published over the past 10 years, and consider our knowledge of the molecular pharmacology of the drug in the context of clinical trial data, attempting to provide a mechanism-guided understanding of its activity.

Genotyping and functional analysis of the D104N variant of human endostatin.

Endostatin is an endogenous inhibitor of angiogenesis derived from the extracellular matrix protein collagen XVIII. It has been reported that a variation at the 104 position (D104N) of human endostatin is associated with an increased risk of prostate cancer, potentially indicating that this protein variant is less active as an anti-angiogenic agent. Herein we reported the results of genotyping 389 patients with androgen independent prostate cancer (AIPC) and 352 normal control individuals for D104N endostatin. There was no significant association between the frequency of 104N endostatin and the incidence of AIPC in either Caucasian or African American patients compared to controls (15% Caucasian AIPC versus 13.7% in Caucasian controls, p=0.79; 7.4% African American AIPC versus 5.6% in African American controls, p=0.64). Actuarial analysis revealed no statistically significant association between incidence of the DN heterozygous genotype and survival (p=0.62 by logrank test). To study the functional significance of the D104N conversion, we have expressed and purified insoluble recombinant human 104D and 104N endostatin and compared their respective activities in human umbilical vein endothelial cell (HUVEC) tube formation assays. The 104N variant of human endostatin inhibited HUVEC tube formation at least as well as the wild-type form. We concluded that the D104N variation in human endostatin is neither clinically relevant nor suitable as a pharmacogenomic endpoint to assess the risk for developing AIPC.

Anti-angiogenic activity of human endostatin is HIF-1-independent in vitro and sensitive to timing of treatment in a human saphenous vein assay.

Endostatin is a 20-kDa endogenous angiogenesis inhibitor that has recently been shown to inhibit the expression of vascular endothelial growth factor (VEGF), an angiogenic growth factor that is up-regulated by hypoxia via the HIF-1 transcription factor complex. To determine if the anti-angiogenic activity of endostatin involves a modulation of the HIF-1/VEGF pathway in cancer cells, experiments were conducted to establish what effect endostatin has on HIF-1 activity, HIF-1alpha protein production, and cellular localization in prostate cancer cells and endothelial cells. Endothelial cell tube formation was inhibited by endostatin purchased from Calbiochem (San Diego, CA) but not endostatin obtained from EntreMed (Rockville, MD). Subsequent experiments using Calbiochem endostatin showed that it did not alter HIF-1alpha protein production or cellular localization in any of the cell lines tested, nor did it alter HIF-1 transactivational activity in hypoxia. Whether or not this is also true in vivo remains to be determined. Nevertheless, these data suggest that the anti-angiogenic activity of endostatin is independent of the HIF-1/VEGF pathway. Immunocytochemical staining results do not indicate a decreased production of VEGF in Calbiochem endostatin-treated LNCaP or human umbilical vein endothelial cells (HUVEC). Treatment of rat aortic cross sections with human endostatin from Calbiochem resulted in a dose-dependent inhibition of microvessel outgrowth. Importantly, inhibition of vessel outgrowth by Calbiochem endostatin in a human saphenous vein angiogenesis assay required early treatment. In view of this in vitro data, we suggest that clinical trials involving endostatin treatment of late-stage disease may not adequately represent the efficacy of this drug in early-stage cancer.

Current status of thalidomide and its role in the treatment of metastatic prostate cancer.

Following the discovery of its anti-angiogenic properties and despite its tragic history, thalidomide has re-surfaced in the field of oncology. Concurrent with its evaluation in various clinical trials for cancer, thalidomide's mechanism of action is sought and new analogues with improved efficacy and pharmacological profile are emerging. This review is a critical evaluation of thalidomide metabolism, molecular targets, anti-angiogenic activity and clinical efficacy with an emphasis on metastatic prostate cancer.

Studies of the biosynthesis of DTX-5a and DTX-5b by the dinoflagellate Prorocentrum maculosum: regiospecificity of the putative Baeyer-Villigerase and insertion of a single amino acid in a polyketide chain.

The biosynthetic origins of the diarrhetic shellfish poisoning toxins DTX-5a and DTX-5b have been elucidated by supplementing cultures of the producing organism Prorocentrum maculosum with stable isotope labeled precursors and determining the incorporation patterns by 13C NMR spectroscopy. The amino acid residue in the sulfated side chain is found to originate from glycine, and oxygen insertion in the chain is shown to occur after polyketide formation.

New directions in cancer research 2003: technological advances in biology, drug resistance, and molecular pharmacology.

The 94th Annual Meeting of the American Association for Cancer Research (AACR) was held from July 11 to 14, 2003 in Washington, DC, and provided an overview of the latest developments in the field of cancer. This report provides highlights of presentations on array-based and RNA-interference technologies to study cancer biology and molecular pharmacology of anticancer drugs, mechanisms and modulation of drug resistance patterns, recent developments in the treatment of prostate cancer, and the medicinal chemistry of established and novel anticancer drugs.

Antiangiogenesis therapeutic strategies in prostate cancer.

It is now well documented that tumor progression from its early stages to an advanced metastatic state requires the recruitment of new vasculature. The reliance on angiogenesis by tumors renders them susceptible to agents that can interfere with the angiogenic process. Recent interest in the therapeutic potential of using angiogenesis as a target mechanism for anticancer therapy has led to the identification of various antiangiogenic agents that interfere at various stages of the process. This review is a summary of recent progress in the identification and characterization of antiangiogenesis agents with a focus on their utility with respect to prostate cancer. Though we focus on prostate cancer, this knowledge is relevant to any cancer that involves angiogenesis.