Junxuan Lü

Selenium-induced inhibition of angiogenesis in mammary cancer at chemopreventive levels of intake

The trace element nutrient selenium (Se) has been shown to possess cancer‐preventive activity in both animal models and humans, but the mechanisms by which this occurs remain to be elucidated. Because angiogenesis is obligatory for the genesis and growth of solid cancers, we investigated, in the study presented here, the hypothesis that Se may exert its cancer‐preventive activity, at least in part, by inhibiting cancer‐associated angiogenesis. The effects of chemopreventive levels of Se on the intra‐tumoral microvessel density and the expression of vascular endothelial growth factor in 1‐methyl‐1‐nitrosourea–induced rat mammary carcinomas and on the proliferation and survival and matrix metalloproteinase activity of human umbilical vein endothelial cells in vitro were examined. Increased Se intake as Se‐enriched garlic, sodium selenite, or Se‐methylselenocysteine led to a significant reduction of intra‐tumoral microvessel density in mammary carcinomas, irrespective of the manner by which Se was provided: continuous exposure (7‐wk feeding) with a chemoprevention protocol or acute bolus exposure (3 d) after carcinomas had established. Compared with the untreated controls, significantly lower levels of vascular endothelial growth factor expression were observed in a sizeable proportion of the Se‐treated carcinomas. In contrast to the mammary carcinomas, the microvessel density of the uninvolved mammary glands was not altered by Se treatment. In cell culture, direct exposure of human umbilical vein endothelial cells to Se induced cell death predominantly through apoptosis, decreased the gelatinolytic activities of matrix metalloproteinase‐2, or both. These results indicate a potential for Se metabolites to inhibit key attributes (proliferation, survival, and matrix degradation) of endothelial cells critical for angiogenic sprouting. Therefore, inhibition of angiogenesis associated with cancer may be a novel mechanism for the anticancer activity of Se in vivo, and multiple mechanisms are probably involved in mediating the anti‐angiogenic activity. Mol. Carcinog. 26:213–226, 1999.

Anti-cancer, anti-diabetic and other pharmacologic and biological activities of penta-galloyl-glucose

Abstract1, 2, 3, 4, 6-penta-O-galloyl-β-D-glucose (PGG) is a polyphenolic compound highly enriched in a number of medicinal herbals. Several in vitro and a handful of in vivo studies have shown that PGG exhibits multiple biological activities which implicate a great potential for PGG in the therapy and prevention of several major diseases including cancer and diabetes. Chemically and functionally, PGG appears to be distinct from its constituent gallic acid or tea polyphenols. For anti-cancer activity, three published in vivo preclinical cancer model studies with PGG support promising efficacy to selectively inhibit malignancy without host toxicity. Potential mechanisms include anti-angiogenesis; anti-proliferative actions through inhibition of DNA replicative synthesis, S-phase arrest, and G1 arrest; induction of apoptosis; anti-inflammation; and anti-oxidation. Putative molecular targets include p53, Stat3, Cox-2, VEGFR1, AP-1, SP-1, Nrf-2, and MMP-9. For anti-diabetic activity, PGG and analogues appear to improve glucose uptake. However, very little is known about the absorption, pharmacokinetics, and metabolism of PGG, or its toxicity profile. The lack of a large quantity of highly pure PGG has been a bottleneck limiting in vivo validation of cancer preventive and therapeutic efficacies in clinically relevant models.

Dissociation of the genotoxic and growth inhibitory effects of selenium

The effects of forms of selenium compounds that enter the cellular selenium metabolic pathway at different points were investigated in a mouse mammary carcinoma cell line. The goal of these experiments was to determine if the genotoxicity of selenium, defined as its ability to induce DNA single-strand breaks, could be dissociated from activities proposed to account for its cancer inhibitory activity. The results demonstrated that growth inhibition, measured as inhibition of cell proliferation and induction of cell death, was induced by all the forms of selenium evaluated. However, sodium selenite and sodium selenide, which are metabolized predominantly to hydrogen selenide, caused the rapid induction of DNA single-strand breaks as an early event that preceded growth inhibition. Interestingly methylselenocyanate and Se-methylselenocysteine, which are initially metabolized predominantly to methylselenol, induced growth inhibition in the absence of DNA single-strand breakage. Differences in the time course of selenium retention, in the occurrence of membrane damage, and in the induction of morphological changes by selenite versus methylselenocyanate were noted. Collectively, these data indicate that different pathways affecting cell proliferation and cell death are induced depending on whether selenium undergoes metabolism predominantly to hydrogen selenide or to methylselenol.

Selenite induction of DNA strand breaks and apoptosis in mouse leukemic L1210 cells

The effects of selenite on DNA integrity, cell viability, and long-term proliferative potential of mouse leukemic L1210 cells were examined in this study. Selenite treatment resulted in concentration-dependent increases in DNA single-strand breaks and double-strand breaks, as detected by a modified filter elution assay. A time-course experiment showed that DNA single-strand breaks preceded DNA double-strand breaks. Agarose gel electrophoresis of DNA extracted from selenite-treated cells displayed a nucleosomal fragmentation pattern that is characteristic of apoptotic cell death. The involvement of a Ca2+,Mg(2+)-dependent endonuclease responsible for DNA double-strand fragmentation was implied by the observation that two inhibitors of endonuclease activity, i.e. aurintricarboxylic acid and zinc, blocked selenite-induced DNA double-strand breaks. These inhibitors also prevented selenite-induced cell death as defined by loss of ability to exclude trypan blue dye. Selenite treatment severely impaired the colony-forming ability of cells capable of trypan blue exclusion. The induction of DNA strand breaks and commitment to apoptosis may explain the selenite-mediated growth inhibition and loss of long-term proliferative potential.

Monomethyl selenium--specific inhibition of MMP-2 and VEGF expression: implications for angiogenic switch regulation.

Previous work suggested that antiangiogenic activity may be a novel mechanism contributing to the cancer chemopreventive activity of selenium (Se). Because methylselenol has been implicated as an in vivo active chemopreventive Se metabolite, experiments were conducted to test the hypothesis that this metabolite pool might inhibit the expression of matrix metalloproteinase‐2 (MMP‐2) by vascular endothelial cells and of vascular endothelial growth factor (VEGF) by cancer epithelial cells, two proteins critical for angiogenesis and its regulation. In human umbilical vein endothelial cells (HUVECs), zymographic analyses showed that short‐term exposure to methylseleninic acid (MSeA) and methylselenocyanate (MSeCN), both immediate methylselenol precursors, decreased the MMP‐2 gelatinolytic activity in a concentration‐dependent manner. In contrast, Se forms that enter the hydrogen selenide pool lacked any inhibitory effect. The methyl Se inhibitory effect on MMP‐2 was cell dependent because direct incubation with Se compounds in the test tube did not result in its inactivation. Immunoblot and enzyme‐linked immunosorbent assay analyses showed that a decrease of the MMP‐2 protein level largely accounted for the methyl Se–induced reduction of gelatinolytic activity. The effect of MSeA on MMP‐2 expression occurred within 0.5 h of exposure and preceded MSeA‐induced reduction of the phosphorylation level of mitogen‐activated protein kinases (MAPKs) 1 and 2 (∼3 h) and endothelial apoptosis (∼25 h). In addition to these biochemical effects in monolayer culture, MSeA and MSeCN exposure decreased HUVEC viability and cell retraction in a three‐dimensional context of capillary tubes formed on Matrigel, whereas comparable or higher concentrations of selenite failed to exert such effects. In human prostate cancer (DU145) and breast cancer (MCF‐7 and MDA‐MB‐468) cell lines, exposure to MSeA but not to selenite led to a rapid and sustained decrease of cellular (lysate) and secreted (conditioned medium) VEGF protein levels irrespective of the serum level (serum‐free medium vs. 10% fetal bovine serum) in which Se treatments were carried out. The concentration of MSeA required for suppressing VEGF expression was much lower than that needed for apoptosis induction. Taken together, the data support the hypothesis that the monomethyl Se pool is a proximal Se for inhibiting the expression of MMP‐2 and VEGF and of angiogenesis. The data also indicate that the methyl Se–specific inhibitory effects on these proteins are rapid and primary actions, preceding or independent of inhibitory effects on mitogenic signaling at the level of MAPK1/2 and on cell growth and survival. Mol. Carcinog. 29:236–250, 2000.

Superior in vivo inhibitory efficacy of methylseleninic acid against human prostate cancer over selenomethionine or selenite

Methylselenol has been implicated as an active anticancer selenium (Se) metabolite. However, its in vivo efficacy against prostate cancer (PCa) has yet to be established. Here, we evaluated the growth inhibitory effects of two presumed methylselenol precursors methylseleninic acid (MSeA) and Se-methylselenocysteine (MSeC) in comparison with selenomethionine (SeMet) and selenite in DU145 and PC-3 human PCa xenografts in athymic nude mice. Each Se was given by a daily single oral dose regimen starting the day after the subcutaneous inoculation of cancer cells. We analyzed serum, liver and tumor Se content to confirm supplementation status and apoptosis indices and tumor microvessel density for association with antitumor efficacy. Furthermore, we analyzed lymphocyte DNA integrity to detect genotoxic effect of Se treatments. The data show that MSeA and MSeC exerted a dose-dependent inhibition of DU145 xenograft growth and both were more potent than SeMet and selenite, in spite of less tumor Se retention than in the SeMet-treated mice. Selenite treatment increased DNA single-strand breaks in peripheral lymphocytes, whereas the other Se forms did not. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) and cleaved caspase-3 indices (apoptosis) from MSeC-treated tumors were higher than tumors from control mice or MSeA-treated mice, whereas the microvessel density index was lower in tumors from MSeA-treated mice. In the PC-3 xenograft model, only MSeA was growth inhibitory at a dose of 3 mg/kg body wt. In summary, our data demonstrated superior in vivo growth inhibitory efficacy of MSeA over SeMet and selenite, against two human PCa xenograft models without the genotoxic property of selenite.

Induction of caspase‐mediated apoptosis and cell‐cycle G1 arrest by selenium metabolite methylselenol

Previous work based on mono‐methyl selenium compounds that are putative precursors of methylselenol has strongly implicated this metabolite in the induction of caspase‐mediated apoptosis of human prostate carcinoma and leukemia cells and G1 arrest in human vascular endothelial and cancer epithelial cells. To test the hypothesis that methylselenol itself is responsible for exerting these cellular effects, we examined the apoptotic action on DU145 human prostate cancer cells and the G1 arrest effect on the human umbilical vein endothelial cells (HUVECs) of methylselenol generated with seleno‐L‐methionine as a substrate for L‐methionine‐α‐deamino‐γ‐mercaptomethane lyase (EC4.4.1.11, also known as methioninase). Exposure of DU145 cells to methylselenol so generated in the sub‐micromolar range led to caspase‐mediated cleavage of poly(ADP‐ribose) polymerase, nucleosomal DNA fragmentation, and morphologic apoptosis and resulted in a profile of biochemical effects similar to that of methylseleninic acid (MSeA) exposure as exemplified by the inhibition of phosphorylation of protein kinase AKT and extracellularly regulated kinases 1/2. In HUVEC, methylselenol exposure recapitulated the G1 arrest action of MSeA in mitogen‐stimulated G1progression during mid‐G1 to late G1. This stage specificity was mimicked by inhibitors of phosphatidylinositol 3‐kinase. The results support methylselenol as an active selenium metabolite for inducing caspase‐mediated apoptosis and cell‐cycle G1 arrest. This cell‐free methylselenol‐generation system is expected to have significant usefulness for studying the biochemical and molecular targeting mechanisms of this critical metabolite and may constitute the basis of a novel therapeutic approach for cancer, using seleno‐L‐methionine as a prodrug.

Methyl-selenium compounds inhibit prostate carcinogenesis in the transgenic adenocarcinoma of mouse prostate model with survival benefit.

Chemoprevention of prostate cancer by second-generation selenium compounds in reference to selenomethionine holds strong promise to deal with the disease at the root. Here we used the transgenic adenocarcinoma mouse prostate (TRAMP) model to establish the efficacy of methylseleninic acid (MSeA) and methylselenocysteine (MSeC) against prostate carcinogenesis and to characterize potential mechanisms. Eight-week-old male TRAMP mice (C57B/6 background) were given a daily oral dose of water, MSeA, or MSeC at 3 mg Se/kg body weight and were euthanized at either 18 or 26 weeks of age. By 18 weeks of age, the genitourinary tract and dorsolateral prostate weights for the MSeA- and MSeC-treated groups were lower than for the control (P < 0.01). At 26 weeks, 4 of 10 control mice had genitourinary weight >2 g, and only 1 of 10 in each of the Se groups did. The efficacy was accompanied by delayed lesion progression, increased apoptosis, and decreased proliferation without appreciable changes of T-antigen expression in the dorsolateral prostate of Se-treated mice and decreased serum insulin-like growth factor I when compared with control mice. In another experiment, giving MSeA to TRAMP mice from 10 or 16 weeks of age increased their survival to 50 weeks of age, and delayed the death due to synaptophysin-positive neuroendocrine carcinomas and synaptophysin-negative prostate lesions and seminal vesicle hypertrophy. Wild-type mice receiving MSeA from 10 weeks did not exhibit decreased body weight or genitourinary weight or increased serum alanine aminotransferase compared with the control mice. Therefore, these selenium compounds may effectively inhibit this model of prostate cancer carcinogenesis.

Differential induction of growth arrest inducible genes by selenium compounds

The effects of two types of selenium compounds on the expression levels of growth arrest and DNA damage-inducible (gadd) genes and on selected cell death genes were examined in mouse mammary MOD cells to test the hypothesis that the diversity of selenium-induced cellular responses to these compounds could be distinguished by unique gene expression patterns. Whereas the expression patterns of known cell death-related genes (bcl-2 and bax) were not informative with respect to the cellular response patterns upon exposure to selenium compounds, time-dependent and selenium species-specific induction patterns were observed for gadd34, gadd45 and gadd153 genes. It was also observed that the MOD cells expressed a truncated p53 transcript but no detectable immunoreactive P53 protein, indicating a null p53 phenotype. The fact that selenium compounds induced growth arrest and death of these cells and that these compounds induced specific patterns of expression of gadd genes indicates that these genes may mediate some selenium-induced cellular responses. The findings further imply that selenium compounds may be effective chemopreventive agents for human breast carcinogenesis, in which p53 mutations are frequent.

Potent Antiandrogen and Androgen Receptor Activities of an Angelica gigas–Containing Herbal Formulation: Identification of Decursin as a Novel and Active Compound with Implications for Prevention and Treatment of Prostate Cancer

Androgen and androgen receptor (AR)-mediated signaling are crucial for the development of prostate cancer. Identification of novel and naturally occurring phytochemicals that target androgen and AR signaling from Oriental medicinal herbs holds exciting promises for the chemoprevention of this disease. In this article, we report the discovery of strong and long-lasting antiandrogen and AR activities of the ethanol extract of a herbal formula (termed KMKKT) containing Korean Angelica gigas Nakai (AGN) root and nine other Oriental herbs in the androgen-dependent LNCaP human prostate cancer cell model. The functional biomarkers evaluated included a suppression of the expression of prostate-specific antigen (PSA) mRNA and protein (IC50, approximately 7 microg/mL, 48-hour exposure) and an inhibition of androgen-induced cell proliferation through G1 arrest and of the ability of androgen to suppress neuroendocrine differentiation at exposure concentrations that did not cause apoptosis. Through activity-guided fractionation, we identified decursin from AGN as a novel antiandrogen and AR compound with an IC50 of approximately 0.4 microg/mL (1.3 micromol/L, 48-hour exposure) for suppressing PSA expression. Decursin also recapitulated the neuroendocrine differentiation induction and G1 arrest actions of the AGN and KMKKT extracts. Mechanistically, decursin in its neat form or as a component of AGN or KMKKT extracts inhibited androgen-stimulated AR translocation to the nucleus and down-regulated AR protein abundance without affecting the AR mRNA level. The novel antiandrogen and AR activities of decursin and decursin-containing herbal extracts have significant implications for the chemoprevention and treatment of prostate cancer and other androgen-dependent diseases.