Hyo-Jeong Lee

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.

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.

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.

Penta-1,2,3,4,6-O-galloyl-β-d-glucose induces p53 and inhibits STAT3 in prostate cancer cells in vitro and suppresses prostate xenograft tumor growth in vivo

Penta-1,2,3,4,6-O-galloyl-β-d-glucose (PGG) is a naturally occurring gallotannin from some Oriental herbs. Several cell culture studies suggested a potential for PGG as a novel agent for the chemoprevention and treatment of cancer. Here, we investigated the cell death signaling mechanisms induced by PGG in human prostate cancer cells of different p53 functional status. We observed the induction of G1- and S-phase arrests and caspase-mediated apoptosis in the androgen-dependent human LNCaP cells, which express wild-type p53, and in the androgen-independent, p53-mutant DU145 cells. In LNCaP cells, caspase-mediated apoptosis induction by PGG was associated with and mediated in major part by activation of p53 as established through small interfering RNA knockdown and dominant-negative mutant approaches. Intracellular reactive oxygen species production by PGG was found to be crucial for these molecular and cellular actions. In DU145 cells, which harbor constitutively active signal transducer and activator of transcription 3 (STAT3), caspase-mediated apoptosis induction by PGG was associated with an inhibition of STAT3 Tyr705 phosphorylation and the down-regulation of STAT3 transcriptional targets Bcl-XL and Mcl-1. Overexpression of Bcl-XL or knockdown of its binding partner Bak attenuated apoptosis induction. Furthermore, we provide, for the first time, in vivo data that PGG significantly inhibited DU145 xenograft growth in an athymic nude mouse model in association with an inhibition of pSTAT3. Our data support PGG as a multitargeting agent for chemoprevention and therapy of prostate cancer by activating the p53 tumor suppressor pathway and by inhibiting STAT3 oncogenic signaling. [Mol Cancer Ther 2008;7(9):2681–91]

Mitochondria-cytochrome C-caspase-9 cascade mediates isorhamnetin-induced apoptosis

Isorhamnetin is a flavanoid present in plants of the Polygonaceae family and is also an immediate metabolite of quercetin in mammals. Since the plasma level of isorhamnetin is maintained longer than quercetin, isorhamnetin may be a key metabolite to mediate the anti-tumor effect of quercetin. In the present study, we investigated the apoptotic mechanism of isorhamnetin in Lewis lung cancer (LLC) cells in vitro and established its in vivo anti-cancer efficacy. In cell culture, isorhamnetin significantly increased DNA fragmentation, and TUNEL positive apoptotic bodies and sub-G(1) apoptotic population in time- and dose-dependent manners. Western blot analyses revealed increased cleavage of caspase-3, and caspase-9 and PARP and increased cytosolic cytochrome C in isorhamnetin-treated cells. These events were accompanied by a reduced mitochondrial potential. Apoptosis was blocked by a general caspase inhibitor or the specific inhibitor of caspase-3 or -9. These in vitro results support mitochondria-dependent caspase activation to mediate isorhamnetin-induced apoptosis. Furthermore, an animal study revealed for the first time that isorhamnetin given by i.p. injection at a dose that is at least one order of magnitude lower than quercetin significantly suppressed the weights of tumors excised from LLC bearing mice. The in vivo anti-tumor efficacy was accompanied by increased TUNEL-positive and cleaved-caspase-3-positive tumor cells. Our data therefore support isorhamnetin as an active anti-cancer metabolite of quercetin in part through caspase-mediated apoptosis.

Coffee phenolic phytochemicals suppress colon cancer metastasis by targeting MEK and TOPK

Epidemiological studies suggest that coffee consumption reduces the risk of cancers, including colon cancer, but the molecular mechanisms and target(s) underlying the chemopreventive effects of coffee and its active ingredient(s) remain unknown. Based on serving size or daily units, coffee contains larger amounts of phenolic phytochemicals than tea or red wine. Coffee or chlorogenic acid inhibited CT-26 colon cancer cell-induced lung metastasis by blocking phosphorylation of ERKs. Coffee or caffeic acid (CaA) strongly suppressed mitogen-activated MEK1 and TOPK activities and bound directly to either MEK1 or TOPK in an ATP-noncompetitive manner. Coffee or CaA, but not caffeine, inhibited ERKs phosphorylation, AP-1 and NF-κB transactivation and subsequently inhibited TPA-, EGF- and H-Ras-induced neoplastic transformation of JB6 P+ cells. Coffee consumption was also associated with a significant attenuation of ERKs phosphorylation in colon cancer patients. These results suggest that coffee and CaA target MEK1 and TOPK to suppress colon cancer metastasis and neoplastic cell transformation.

Sphingosine kinase 1 pathway is involved in melatonin-induced HIF-1α inactivation in hypoxic PC-3 prostate cancer cells

Abstract:  Sphingosine kinase 1 (SPHK1) is a newly discovered modulator of hypoxia inducible factor 1α (HIF‐1α) with various biological activities such as cell growth, survival, invasion, angiogenesis, and carcinogenesis. Thus, in the present study, the biological mechanisms of melatonin were elucidated in association with SPHK1 pathway in PC‐3 prostate cancer cells under hypoxia. Melatonin inhibited the stability of HIF‐1α in a time‐ and concentration‐ dependent manners. Also, melatonin decreased SPHK1 activity in PC‐3 cells during hypoxia. Furthermore, melatonin suppressed AKT/glycogen synthase kinase‐3β (GSK‐3β) signaling pathway, which stabilizes HIF‐1α via inhibition of von Hippel‐Lindau tumor suppressor protein. Consistently, siRNA‐SPHK1 and sphingosine kinase inhibitor (SKI) effectively blocked the expression of HIF‐1α, phospho‐AKT and vascular endothelial growth factor (VEGF) production in PC‐3 cells under hypoxia, suggesting the role of SPHK1 in melatonin‐inhibited HIF‐1α accumulation. Moreover, reactive oxygen species (ROS) scavenger N‐acteylcysteine enhanced melatonin‐inhibited HIF‐1α expression and SPHK1 activity. Overall, our findings suggest that melatonin suppresses HIF‐1α accumulation via inhibition of SPHK1 pathway and ROS generation in PC‐3 cells under hypoxia.

Suppression of STAT3 and HIF-1 alpha mediates anti-angiogenic activity of betulinic acid in hypoxic PC-3 prostate cancer cells.

Background Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that regulates various cellular processes such as cell survival, angiogenesis and proliferation. In the present study, we examined that betulinic acid (BA), a triterpene from the bark of white birch, had the inhibitory effects on hypoxia-mediated activation of STAT3 in androgen independent human prostate cancer PC-3 cells. Methodology/Principal Findings BA inhibited the protein expression and the transcriptional activities of hypoxia-inducible factor-1α (HIF-1α) under hypoxic condition. Consistently, BA blocked hypoxia-induced phosphorylation, DNA binding activity and nuclear accumulation of STAT3. In addition, BA significantly reduced cellular and secreted levels of vascular endothelial growth factor (VEGF), a critical angiogenic factor and a target gene of STAT3 induced under hypoxia. Furthermore, BA prevented in vitro capillary tube formation in human umbilical vein endothelial cells (HUVECs) maintained in conditioned medium of hypoxic PC-3 cells, implying anti-angiogenic activity of BA under hypoxic condition. Of note, chromatin immunoprecipitation (ChiP) assay revealed that BA inhibited binding of HIF-1α and STAT3 to VEGF promoter. Furthermore, silencing STAT3 using siRNA transfection effectively enhanced the reduced VEGF production induced by BA treatment under hypoxia. Conclusions/Significance Taken together, our results suggest that BA has anti-angiogenic activity by disturbing the binding of HIF-1α and STAT3 to the VEGF promoter in hypoxic PC-3 cells.

Cyclooxygenase-2/prostaglandin E2 pathway mediates icariside II induced apoptosis in human PC-3 prostate cancer cells

Icariside II (IS) isolated from the roots of Epimedium koreanum Nakai was known to have antioxidant activity and inhibit melanogenesis and hypoxia inducible factor. We report here for the first time that IS induces apoptosis through its anti-inflammatory effects in PC-3 prostate cancer cells. IS exerted cytotoxicity against PC-3 cells with IC(50) of approximately 20 microM. IS suppressed both constitutive and arachidonic acid (AA)-induced cyclooxygenase-2 (COX-2) expression as well as reduced prostaglandin E2 (PGE2) levels in PC-3 cells even at a low concentrations (5 and 10 microM). Additionally, IS increased sub G1 apoptotic portion and exhibited terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL)-positive apoptotic bodies in PC-3 cells at higher concentrations (20 and 40 microM). Furthermore, IS attenuated the mitochondrial membrane potential, released cytochrome C into cytosol, activated caspase-9, -8, and -3 expressions and cleaved poly (ADP-ribose) polymerase (PARP) in PC-3 cells. Consistently, COX-2, inducible NO synthase (iNOS), and vascular endothelial growth factor (VEGF) expressions were suppressed while in parallel inducing apoptosis in hormone-independent prostate carcinoma cells PC-3. Moreover, exogeneous PGE2 inhibited IS induced PARP cleavage in PC-3 cells and also knockdown of COX-2 by siRNA potentiated IS induced PARP cleavage, thereby implicating the critical role of COX-2 pathway in IS induced apoptosis. Taken together, these findings demonstrate that IS initiates the inhibition of COX-2/PGE(2) pathway and then induces apoptosis mainly via mitochondrial dependent pathway in PC-3 prostate cancer cells as a potent cancer chemotherapeutic agent.

A novel class of pyranocoumarin anti–androgen receptor signaling compounds

Androgen and the androgen receptor (AR)–mediated signaling are crucial for prostate cancer development. Novel agents that can inhibit AR signaling in ligand-dependent and ligand-independent manners are desirable for the chemoprevention of prostate carcinogenesis and for the treatment of advanced prostate cancer. We have shown recently that the pyranocoumarin compound decursin from the herb Angelica gigas possesses potent anti-AR activities distinct from the anti–androgen bicalutamide. Here, we compared the anti-AR activities and the cell cycle arrest and apoptotic effects of decursin and two natural analogues in the androgen-dependent LNCaP human prostate cancer cell culture model to identify structure-activity relationships and mechanisms. Decursin and its isomer decursinol angelate decreased prostate-specific antigen expression with IC50 of ∼1 μmol/L. Both inhibited the androgen-stimulated AR nuclear translocation and transactivation, decreased AR protein abundance through proteasomal degradation, and induced G0/1 arrest and morphologic differentiation. They also induced caspase-mediated apoptosis and reactive oxygen species at higher concentrations. Furthermore, they lacked the agonist activity of bicalutamide in the absence of androgen and were more potent than bicalutamide for suppressing androgen-stimulated cell growth. Decursinol, which does not contain a side chain, lacked the reactive oxygen species induction and apoptotic activities and exerted paradoxically an inhibitory and a stimulatory effect on AR signaling and cell growth. In conclusion, decursin and decursinol angelate are members of a novel class of nonsteroidal compounds that exert a long-lasting inhibition of both ligand-dependent and ligand-independent AR signaling. The side chain is critical for sustaining the anti-AR activities and the growth arrest and apoptotic effects. [Mol Cancer Ther 2007;6(3):907–17]