Donald L. Hill

In vitro anti-cancer activity and structure-activity relationships of natural products isolated from fruits of Panax ginseng.

PurposePanax ginseng and its extracts have long been used for medical purposes; there is increasing interest in developing ginseng products as cancer preventive or therapeutic agents. The present study was designed to determine biological structure–activity relationships (SAR) for saponins present in Panax ginseng fruits.MethodsEleven saponins were extracted from P. ginseng fruits and purified by use of D101 resin and ordinary and reverse-phase silica gel column chromatography. Their chemical structures were elucidated on the basis of physicochemical constants and NMR spectra. Compounds were then evaluated for SAR with their in vitro cytotoxicity against several human cancer cell lines.ResultsThe 11 compounds were identified as 20(R)-dammarane-3β,12β,20,25-tetrol (25-OH-PPD, 1); 20(R)-dammarane-3β,6α,12β,20,25-pentol (25-OH-PPT, 2); 20(S)-protopanaxadiol (PPD, 3); daucosterine 4, 20(S)-ginsenoside-Rh2 (Rh2, 5); 20(S)-ginsenoside-Rg3 (Rg3,6); 20(S)-ginsenoside-Rg2 (Rg2, 7); 20(S)-ginsenoside-Rg1 (Rg1, 8); 20(S)-ginsenoside-Rd (Rd, 9); 20(S)-ginsenoside-Re (Re, 10); and 20(S)-ginsenoside-Rb1 (Rb1, 11). Among the eleven compounds, 1, 3 and 5 were the most effective inhibitors of cell growth and proliferation and inducers of apoptosis and cell cycle arrest. For 1, the IC50 values for most cell lines were in the range of 10–60xa0μM, at least twofold lower than for any of the other compounds. Compounds 1 and 3 had significant, dose-dependent effects on apoptosis, proliferation, and cell cycle progression.ConclusionsThe results suggest that the type of dammarane, the number of sugar moieties, and differences in the substituent groups affect their anti-cancer activity. This information may be useful for evaluating the structure/function relationship of other ginsenosides and their aglycones and for development of novel anticancer agents.

Ribosomal protein S7 as a novel modulator of p53–MDM2 interaction: binding to MDM2, stabilization of p53 protein, and activation of p53 function

As a major negative regulator of p53, the MDM2 oncogene plays an important role in carcinogenesis and tumor progression. MDM2 promotes p53 proteasomal degradation and negatively regulates p53 function. The mechanisms by which the MDM2–p53 interaction is regulated are not fully understood, although several MDM2-interacting molecules have recently been identified. To search for novel MDM2-binding partners, we screened a human prostate cDNA library by the yeast two-hybrid assay using full-length MDM2 protein as the bait. Among the candidate proteins, ribosomal protein S7 was identified and confirmed as a novel MDM2–interacting protein. Herein, we demonstrate that S7 binds to MDM2, in vitro and in vivo, and that the interaction between MDM2 and S7 leads to modulation of MDM2-p53 binding by forming a ternary complex among MDM2, p53 and S7. This results in the stabilization of p53 protein through abrogation of MDM2-mediated p53 ubiquitination. Consequently, S7 overexpression increases p53 transactivational activities, induces apoptosis, and inhibits cell proliferation. The identification of S7 as a novel MDM2-interacting partner contributes to elucidation of the complex regulation of the MDM2–p53 interaction and has implications in cancer prevention and therapy.

Genistein, a Dietary Isoflavone, Down-Regulates the MDM2 Oncogene at Both Transcriptional and Posttranslational Levels

Although genistein has chemopreventive effects in several human malignancies, including cancers of the breast, colon, and prostate, the mechanisms of action are not fully understood. Herein we report novel mechanisms whereby genistein down-regulates the MDM2 oncogene, perhaps explaining some of its anticancer activities. In a dose- and time-dependent manner, genistein reduced MDM2 protein and mRNA levels in human cell lines of breast, colon, and prostate cancer; primary fibroblasts; and breast epithelial cells. The inhibitory effects were found at both transcriptional and posttranslational levels and were independent of tyrosine kinase pathways. We found that the NFAT transcription site in the region between -132 and +33 in the MDM2 P2 promoter was responsive to genistein. At the posttranslational level, genistein induced ubiquitination of MDM2, which led to its degradation. Additionally, genistein induced apoptosis and G2 arrest and inhibited proliferation in a variety of human cancer cell lines, regardless of p53 status. We further showed that MDM2 overexpression abrogated genistein-induced apoptosis in vitro and that genistein inhibited MDM2 expression and tumor growth in PC3 xenografts. In conclusion, genistein directly down-regulates the MDM2 oncogene, representing a novel mechanism of its action that may have implications for its chemopreventive and chemotherapeutic effects.

Experimental therapy of prostate cancer with novel natural product anti-cancer ginsenosides.

Ginseng and its components exert various biological effects, including antioxidant, anti‐carcinogenic, anti‐mutagenic, and anti‐tumor activity, and recent research has focused on their value in human cancer prevention and treatment. We recently isolated 25‐hydroxyprotopanaxadiol (25‐OH‐PPD) and 25‐hydroxyprotopanaxatriol (25‐OH‐PPT) from Panax ginseng and evaluated their anti‐cancer activity in vitro.

9cUAB30, an RXR specific retinoid, and/or tamoxifen in the prevention of methylnitrosourea-induced mammary cancers

Studies were performed in female Sprague-Dawley rats to determine the efficacy of a new RXR specific retinoid (9cUAB30) when combined with tamoxifen in the prevention of mammary cancers and to determine various pharmacokinetic parameters of the retinoid. When administered by gavage, 9cUAB30 was rapidly absorbed and had a serum t(1/2) of 13.5 h. Since the retinoid was administered in the diet for the chemoprevention study, a 28-day study in which 9cUAB30 was given at dose levels of 200, 400, and 600 mg/kg diet revealed fairly constant serum levels regardless of dose or length of treatment; possibly accounting for the observed low toxicity of this compound. When suboptimal doses of 9cUAB30 were given in the methylnitrosourea (MNU)-induced mammary cancer model, the following average number of mammary cancers were observed: 9cUAB30 (150 mg/kg diet), 4.3; tamoxifen (0.4 mg/kg diet), 4.6; 9cUAB30 (150 mg/kg diet)+tamoxifen (0.4 mg/kg diet), 2.6; and controls, 6.0. Thus, the combination of the agents resulted in an increased effect in preventing mammary cancers; suggesting that cancer cell proliferation was inhibited by the compounds blocking different pathways.

Radiolabeled 2′-fluorodeoxyuracil-β-D-arabinofuranoside (FAU) and 2′-fluoro-5-methyldeoxyuracil-β-D-arabinofuranoside (FMAU) as tumor-imaging agents in mice

Abstract.Purpose: The purpose of the present study was to evaluate, in conjunction with the National Cancer Institute, the feasibility of using two thymidine analogs, 2′-fluorodeoxyuracil-β-D-arabinofuranoside (FAU, NSC-678515) and 2′-fluoro-5-methyldeoxyuracil-β-D-arabinofuranoside (FMAU, NSC-678516), as 18-fluorine-labeled positron emission tomography (PET) imaging agents. Methods: The in vivo distribution and DNA incorporation of [2-14C]FAU, [2-14C]FMAU, and [2-14C]thymidine (as a control) were studied in SCID mice bearing human xenografts of T-cell leukemia CCRF-CEM. Levels of drug-associated radioactivity in blood, tumor and normal tissues including liver, kidneys, heart, lungs, spleen, brain, and skeletal muscle were determined. Results: At 1xa0h after dosing, radioactivity from all three compounds was distributed in a generally nonspecific manner, except that spleen and tumor tissue had relatively high concentrations of radioactivity from [14C]thymidine. At 4xa0h after dosing, the concentrations of radioactivity from [14C]thymidine and [14C]FMAU were relatively high in spleen and tumor tissue, and that from [14C]FAU was highest in tumor tissue. The tumor/skeletal muscle concentration ratios were 2.25±0.69 and 3.07±0.42 for [14C]FAU and [14C]FMAU, respectively. At 24xa0h after dosing, only spleen and tumor tissues contained appreciable amounts of radioactivity from either compound. In tumor tissue, the levels of radioactivity from [14C]FMAU were two- to threefold greater than those from [14C]thymidine or [14C]FAU. Examination of purified genomic DNA from tumor, liver, kidneys, brain, and skeletal muscle showed that, at 24xa0h after dosing, only DNA from tumor tissue contained appreciable concentrations of radioactivity. Radioactivity from [14C]FMAU in tumor DNA was 45% greater than that from [14C]thymidine and about threefold greater than that from [14C]FAU. Conclusions: The extent of accumulation of [14C]FMAU in tumor tissue and incorporation into tumor DNA indicate that [18F]FMAU could be useful as a functional PET tumor-imaging agent.

Oncogenes as novel targets for cancer therapy (part I): growth factors and protein tyrosine kinases.

In the past 10 years, progress made in cancer biology, genetics, and biotechnology has led to a major transition in cancer drug design and development. There has been a change from an emphasis on non-specific, cytotoxic agents to specific, molecular-based therapeutics.Mechanism-based therapy is designed to act on cellular and molecular targets that are causally involved in the formation, growth, and progression of human cancers. These agents, which may have greater selectivity for cancer versus normal cells, and which may produce better anti-tumor efficacy and lower host toxicity, can be small molecules, natural or engineered peptides, proteins, antibodies, or synthetic nucleic acids (e.g. antisense oligonucleotides, ribozymes, and siRNAs). Novel targets are identified and validated by state-of-the-art approaches, including high-throughput screening, combinatorial chemistry, and gene expression arrays, which increase the speed and efficiency of drug discovery and development. Examples of oncogene-based, molecular therapeutics that show promising clinical activity include trastuzumab (Herceptin®), imatinib (Gleevec®), and gefitinib (Iressa®).However, the full potential of oncogenes as novel targets for cancer therapy has not been realized and many challenges remain, from the validation of novel targets, to the design of specific agents, to the evaluation of these agents in both preclinical and clinical settings. In maximizing the benefits of molecular therapeutics in monotherapy or combination therapy of cancer, it is necessary to have an understanding of the underlying molecular abnormalities and mechanisms involved.This is the first part of a four-part review in which we discuss progress made in the last decade as it relates to the discovery of novel oncogenes and signal transduction pathways, in the context of their potential as targets for cancer therapy. This part delineates the latest discoveries about the potential use of growth factors and protein tyrosine kinases as targets for therapy. Later parts focus on intermediate signaling pathways, transcription factors, and proteins involved in cell cycle, DNA damage, and apoptotic pathways.

Acetylation of Aurora B by TIP60 ensures accurate chromosomal segregation

Faithful chromosome segregation in mammalian cells requires the bi-orientation of sister chromatids which relies on sensing correct attachments between spindle microtubules and kinetochores. Although the mechanisms underlying cyclin-dependent kinase 1 (CDK1) activation that triggers mitotic entry is extensively studied, the regulatory mechanisms that couple CDK1-cyclin B activity to chromosome stability are not well understood. Here, we identified a signaling axis in which Aurora B activity is modulated by CDK1-cyclin B via acetyltransferase TIP60 (Tat-interactive protein 60 kDa) in human cell division. CDK1-cyclin B phosphorylated Ser90 of TIP60, which elicited TIP60-dependent acetylation of Aurora B and promoted accurate chromosome segregation in mitosis. Mechanistically, TIP60 acetylation of Aurora B at Lys215 protected the phosphorylation of its activation loop from PP2A reactivation-elicited dephosphorylation to ensure a robust, error-free metaphase-anaphase transition. These findings delineated a conserved signaling cascade that integrates protein phosphorylation and acetylation to cell cycle progression for maintenance of genomic stability.

Dynamic localization of Mps1 kinase to kinetochores is essential for accurate spindle microtubule attachment

Significance The spindle assembly checkpoint (SAC) works as a surveillance mechanism to ensure accurate segregation of genetic materials during cell division. Protein kinase monopolar spindle 1 (Mps1) plays a key role in SAC, but the mechanism of Mps1 action in chromosome segregation remains elusive. In this study, we identified a previously undefined structural determinant of Mps1 named “IRK” (internal region for kinetochore localization) and demonstrated its functional importance in accurate kinetochore–microtubule attachment. Mechanistically, a dynamic hierarchical interaction between Mps1 and the nuclear division cycle 80 complex (Ndc80C) orchestrates accurate mitosis, because persistent association of inactive Mps1 with Ndc80C via the IRK perturbs correct kinetochore–microtubule attachment. Our results provide a new mechanistic insight into the spatiotemporal dynamics of Mps1 activity at the kinetochore in mitosis. The spindle assembly checkpoint (SAC) is a conserved signaling pathway that monitors faithful chromosome segregation during mitosis. As a core component of SAC, the evolutionarily conserved kinase monopolar spindle 1 (Mps1) has been implicated in regulating chromosome alignment, but the underlying molecular mechanism remains unclear. Our molecular delineation of Mps1 activity in SAC led to discovery of a previously unidentified structural determinant underlying Mps1 function at the kinetochores. Here, we show that Mps1 contains an internal region for kinetochore localization (IRK) adjacent to the tetratricopeptide repeat domain. Importantly, the IRK region determines the kinetochore localization of inactive Mps1, and an accumulation of inactive Mps1 perturbs accurate chromosome alignment and mitotic progression. Mechanistically, the IRK region binds to the nuclear division cycle 80 complex (Ndc80C), and accumulation of inactive Mps1 at the kinetochores prevents a dynamic interaction between Ndc80C and spindle microtubules (MTs), resulting in an aberrant kinetochore attachment. Thus, our results present a previously undefined mechanism by which Mps1 functions in chromosome alignment by orchestrating Ndc80C–MT interactions and highlight the importance of the precise spatiotemporal regulation of Mps1 kinase activity and kinetochore localization in accurate mitotic progression.

Retinoic acid (RA) receptor transcriptional activation correlates with inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase (ODC) activity by retinoids: a potential role for trans-RA-induced ZBP-89 in ODC inhibition.

Evaluation of retinoic acid receptor (RAR) subtype–selective α and γ agonists and antagonists and a retinoid X receptor (RXR) class–selective agonist for efficacy at inhibiting both induction of ornithine decarboxylase (ODC) by the tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in mouse epidermis and rat tracheal epithelial cells and the appearance of papillomas in mouse epidermis treated in the 2‐stage tumor initiation–promotion model indicated that (i) RXR class–selective transcriptional agonists, such as MM11246, were not involved in ODC inhibition; (ii) RAR‐selective agonists that induce gene transcription from RA‐responsive elements (RAREs) were active at low concentrations; (iii) RAR‐selective antagonists that bind RARs and inhibit AP‐1 activation on the collagenase promoter but do not activate RAREs to induce gene transcription were less effective inhibitors; and (iv) RARγ‐selective retinoid agonists were more effective inhibitors of TPA‐induced ODC activity than RARα‐selective agonists. These results suggest that RARE activation has a more important role in inhibition of ODC activity than RXR activation or AP‐1 inhibition and that RARγ‐selective agonists would be the most useful inhibitors of epithelial cell proliferation induced by tumor promoters. The natural retinoid all‐trans‐RA induced expression of transcription factor ZBP‐89, which represses activation of the GC box in the ODC promoter by the transcription factor Sp1. Int. J. Cancer 91:8–21, 2001.